KR0133544B1 - Human , , t cell antigen receptor polypeptides and nucleic - Google Patents

Abstract

The present invention relates to a human γ T cell antigen receptor polypeptide type called type 2bc, which consists of a portion comprising only four sequences encoded in two Cγ CII exon copies. The invention also relates to T cell antigen receptor heterodimers consisting of 2bc γ polypeptides and nucleic acids encoding type 2bc. It also relates to a method of making expression of a γδ T cell antigen receptor heterodimer. The present invention also relates to monoclonal antibodies that specifically react with ephetopes of γ, γ polypeptides. In certain embodiments, such antibodies are reactive with a portion of delta, a variable portion of delta, or a gamma portion. Such antibodies can be identified by collaboration with the CD3 antigen.

Description

Method for producing polypeptite heterodimer that reacts with human T cells

The T cell antigen receptor (TCR) has been shown to be a clone specific disulfide-bonded heterodimer in T cells, consisting of two glycosylated western units, one of which is referred to as α chain and the other to β chain This is called. α and β TCR subunits have a relative molecular weight (M _r ) of approximately 50,000 and 40,000 Daltons, respectively (Allison et al., 1982, Immunol. 129: 2293-2300; Meuer et al., 1983, J. Exp. Med). 157: 705-719; Haskins et al., 1983, J. Exp. Med. 157: 1149-1169). Rearranged during T cell development and βTCR (Yanagi et al., 1984, Nature 308: 145-149; Hedrick et al., 1984, Nature 308: 153-159) and αTCR (Chien et al., 1984, Natuer 312). Saito et al., 1984, Nature 312: 36-40, Sim et al., 1984, Nature 312: 771-775), or by alternative hybridization of a gene encoding a subunit Isolated by probe with nucleotides.

The α and β chains of the T cell claw T cell antigen receptor consist of unique binding of regions termed variable (V), diversity (D), linkage (J) and constant (C), respectively (Siu et al., 1984 , Cell 37: 393; Yanagi et al., 1985, Proc Natl. Acad. Sci. USA 82: 3430). Hypervarable regions were identified (Patten at al., 1984 Neture 312: 40; Becker et. al., 1985, Nature 317: 430). The binding of both V, D and J regions of both α and β chains in each T cell clone is involved in antigen recognition by defining unique binding sites that are unique to the T cell clone and also known as the genotype of the T cell clone. do. In contrast, the C region is not involved in antigen binding.

The unique features of human α, βTCR are the CD3 glycoprotein complexes observed and co-regulation of α, βTCR (Meuer et al., 1983, J. Exp. Med. 157: 705-719), and co-immunoprecipitation (PCT International No. WO 88/00209, published January 14, 1988; Oettgen, et al., 1984 J. Biol. Chem. 259: 12,039-12,048) and co-expression of the disturbances (Weiss et al., 1984, J. Exp. Med. 160 1284-1299). Thus, a direct physical association of two protein complexes is shown by chemically crosslinking the α, βTCR molecule to this T3 glycoprotein and identifying the components of the crosslinked complex as a TCR subunit and a T3 glycoprotein (M _r 28,000) subunit. (Brenner et al., 1984 Cell 40: 183-190). T3 counterparts were similarly associated with α, β TCR in mice (Allison et al., 1985 Nature 314: 107-109; Samelson et al., Immunol. Rev. 81: 131-144).

A third gene, called λTCR and rearranged in T cells, was initially identified in mice (Saito et al., 1984, Natur 309: 757-762; Kranz et al., 1985, Natur 313: 762-755; Hayday et al., 1985, Cell 40: 259-269) and then in humans (Lefranc et al., 1985, Natur 316: 464-466; Murre et al., 1985, Natur 316: 549-522). The human γTCR position appears to be composed of a binding of 5 and a constant region gene of 2 between 5 and 10 variables (Dialyans et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83: 2691). Although the total number of functional variables and linkage regions is limited, significant diversity has been inserted during VJ's linkage process (Kranz et al., 1985, Natur 313: 752-755; Lefranc et al., 1986, Cell 45: 237-246). Quertermaus et al., 1986, Natur 322: 184. γ TCR gene rearrangement occurs not only in inhibitor-cytotoxicity but also in adjuvant phenotype (Lefranc et al., 1985, Natuer 316: 464-466; Murre et al., 1985, Natuer 316: 549-552, Quertermaus et al., 1986, Science 213: 252-255; Lefranc et al., 1986, Cell 45: 237-246, Iwamoto et al., 1986, J. Exp. Med. 163: 1203-1212; Zauderer et al., 1986, J Exp. Med. 163: 1314-1318).

The product of γ TCR was identified in T3 co-precipitates from α, β TCR ^- CD3 ⁺ T (Brenner et al., 1986, Natuer 322: 145-149; Bank et al., 1986, Nature 322: 181; Borst et al. , 1987, Nature 325, 683-688; Moingeon et al., 1987, Nature 325,723-726, PCT International Publication No. WO 88/00209, pub-lished Janauary 14, 1987). γTCR polyfeltide was identified using monoclonal antibodies directed against the γTCR peptide sequence, which polypeptide was found to be inserted into a heterodimer with another polypeptide called δTCR (Brenner et al., Nature 322: 145-149). γ, δ heterodimers have been reported to be non-covalently associated with CD3.

The use of antiserum intended to combat γTCR-specific peptides has led to the identification of CD3-associated γTCR polypeptides in cells derived from peripheral blood, pleural and leukemia cell cultures (Brenner et al., 1986, Nature 322: 145-149). Bank et al., 1986, Nature 322: 179-181, Weiss et al., 1986., Proc. Natl. Acad.Sci USA 83: 6998-7002, Brenner et al., 1987, Nature 325: 689-694 Lew et al., 1986, Science 234: 1401-1405). Bank et al. (Above) disclose a 44KD γ type associated with 62,000KD peptide and T3 on the surface of human thymic cell clones. Similar γTCR polypeptides have also been identified in T lymphocytes of rats, and expression of these peptides during thymic cell sorting is the subject of modern research (Roulet et al., 1985, Nature 314: 103-107; Snodgrass et al., 1985, Nature 315: 232-233; Lew et al., 1986, Science 234: 1401-1408; Pardau et al., 1987, Nature 326: 79-81; Bluesstone et al., 1987, Nature 326: 82-84).

γTCR ⁺ human cell cultures have been studied to confirm that two different γTCR polypeptides have different molecular weights and the ability to form disulfide bonds (Borst et al., 1987, Nature 325: 683-688; Brennet et al. , 1987, Nature 325: 689-694; Moingeon et al., 1987, Nature 325: 723-726; Lanier et al., 1987, J. Exp Med. 165: 1076). Two different γTCR constant region gene fragments, called Cγ1 and Cγ2, respectively, were compared. Cysteine residues encoded by the second exon of Cγ1 were not present in the Cγ2 exon fragment and were therefore proposed to explain the inability of some γTCR peptides to form disulfide bonds (Krangerl et al., 1987, Seience, 237: 1051). -1055; Littman et al., Nature 326: 85088).

Contrary to the polymorphism of γTCR, it appears that only the δTCR constant region is relatively invariant with δ TCR molecules (Hata et al., 1987, Science 238: 678-682).

ΓTCRT gene rearrangement progresses β and αTCR gene rearrangement during T cell oncogenesis (Roulet et al., 1985, Nature 314: 103-107; Snodgrass et al., 1985, Nature 315: 232-233; Sangoter et al., 1986, J. Exp. Med. 163-1491-1508).

Relatively small part from T cell to an aging cycle is γ δTCR, and CD3 ⁺ 4 ^{- +} 8 indicates whether which one of the surface antigens. CD3 ⁺ 4 ^- 8 ^- T cells constitutes approximately 2% of aging CD3 ⁺ T cells. Most fermentation CD3 ⁺ 4 ⁺ or CD3 ⁺ 8 ⁺ key tissue compatible location (MHC) in a restricted cytotoxic T cell, as opposed but the CD3 + natural killer cells, similarly γδTCR ⁺ CD3 ⁺ 4 ^- 8 ^- of the kulron lymphocytes is MHC non-showed to exhibit limited cell degradation activity, but unlike a natural killer cell such ^{γδ + CD3 + 4 - 8 -} T cells were being killed consistently natural killer cell targets, such as K-562 (Borst et al., 1987, 325: 683-688; Brenner et al., 1987, Nature 325: 689-694; Moingeon et al., 1987, Nature 325: 723-726; Blueston et al., 1987, Nature 326: 82-84).

Summary of the Invention

The present invention relates to a form of human γT cell antigen receptor (TCR) polypeptide called Form 2bc. Form 2bc γTCR chains have a first amino acid sequence, as shown substantially in FIG. Form 2bc γTCR chain has a molecular weight of about 40,000 Daltons and comprises a constant region containing the sequence encoded only by two Cγ2 CII exon transcriptions. The invention also relates to a TCR heterodimer comprising γTCR polypeptide form 2bc.

The present invention relates to a nucleic acid sequence comprising a Cγ2 constant region having only two CII exons in a nucleic acid sequence encoding γTCR form 2bc. In a specific embodiment the nucleic acid of the invention comprises at least one portion of the nucleic acid sequence shown in FIG.

The present invention also provides monoclonal antibodies that specifically react with epitopes of γ or δ TCR polypeptides. Such antibodies can be identified by detecting the ability to co-regulate CD3 antigen in cells expressing both γδ TCR and CD3 antigen. In one specific embodiment the invention relates to antibodies that react with the variable region of the δ TCR chain. In particular embodiments such antibodies can be used to detect functional δTCR variable gene rearrangement in a cell. In another embodiment the invention can be used to detect δ TCR variable gene rearrangement. In another embodiment the invention relates to antibodies that react with a certain region of the δTCR polypeptide. In another embodiment the invention is directed to antibodies that react with certain regions of a γTCR polypeptide.

Another feature of the invention provides a method of making expression of γδ TCR in a cell.

3.1 Definition

As used herein, the following terms have the following meanings:

TCR = T cell antigen receptor

V = variable

D = diversity

J = Connectivity

C = constant

mAb = monoclonal antibody.

The present invention relates to a form of human γ T cell antigen receptor polypeptide called form 2bc having a molecular weight of about 40,000 Daltons and comprising a sequence encoded only by two Cγ CII exon transcriptions in a constant region thereof. The invention also relates to T cell antigen receptor heterodimers comprising γ form 2bc and to nucleic acid sequences that encode γ form 2bc and their sites. The present invention also provides monoclonal antibodies that specifically react with epitopes of γ or δT cell antigen receptor polypeptides.

FIG. 1 is a cytopulograph analysis of T cell cultures with anti-TCRδ1.

2 is an immunochemical analysis of the specificity of mAb anti-TCRδ. IDP2 cells labeled I-labeled on the surface were treated with reference mAb P3 (columns 1 and 2), anti-leu 4 (columns 3-5), anti-TCR (columns 6-9) or anti-Cγ serum (column 9) Was immunoprecipitated and then dissolved by SDS-PAGE (polyacrylamide gel electrophoresis) and visualized by radiographic method. N = non-reducing condition; R = reduction condition.

3 shows N-glycanas digestion of δ TCR.

4 is an explanatory diagram of pGEM3-0-240 / 38.

5 shows immunoprecipitation of in vitro translation products of cDNA clone IDP2 0-240 / 38 by mAb anti-TCRδ.

6 shows immunoprecipitation and SDS-PAGE analysis of T cell antigen receptor. The open arrow indicates the position of the δ chain. Continuous arrows indicate the position of the γ chain. Lysates were immunoprecipitated using the δ TCAR-3 antibody (base sequence) or βF1 antibody (excellent sequence).

7 shows immunoprecipitation of the δ chain by the δ TCAR-3 antibody. MOLT-13 cells were lysed in Tris-buffered saline (pH8) (column 1) or 1% Triton X-100 (columns 2-7) containing 0.3% CHAPS (column 1). Column 1 immunoprecipitates γ, δTCR heterodimer with δTCAR-3 having the CD3 protein. Columns 3 and 4 immunoprecipitate a single δ chain from each of the modified lysates (N) and reducing (R) conditions of δTCR-3. Row 5 shows that UCHT-1 immunoprecipitates CD3 protein. Column 6 does not allow the βF1 antibody to immunoprecipitate heterodimers from MOLT-13 cells. Column 7 anti-Cγ antiserum immunoprecipitates a single γ chain.

8 shows the analysis of cell surfaces stained by flow cytometry. γ, δ TCR-positive cells (MOLT-13) PEER, IDP2) and α, βTCR-positive cells (HPB-ALL, Jurkat) were incubated with δTCAR-3, OKT3, WT31 and normal blood serum (NMS) antibodies and flow cytometry Analyzed. B cell culture Daudi was negative.

Figure 9 shows two-color cytofluorograph analysis of δTCAR-3 ⁺ and OKT3 ⁺ peripheral blood lymphocytes, fluorescence isothiocyanate (FITC) fluorescence on the Y axis and phycoerythrin (PE) fluorescence on the X axis. Indicates. The CD3 ⁺ ,, γ, δ TCR ⁺ cells of this sample show 2.4% CD3 ⁺ lymphocytes.

10 shows the measurement of Ca ²⁺ concentration ([Ca ²⁺ ] i) in the cytoplasm over time. Top panel: δ TCAR-3, bottom panel: anti-Leu antibody, arrow indicates time of antibody addition.

Figure 11 shows three types of immunoprecipitation of γ, δTCAR, and the antibodies used for immunoprecipitation for the AE portion are anti-Leu 4 (anti-CD3), βF1 (anti-TCRβ), anti-δ1TCR (anti-δTCR), anti -Cγb serum (anti-γTCR) and P3 (unlabeled fever, reference). Immunoprecipitates from ¹²⁵ I-labeled cytolysis products were analyzed by SDS-PAGE (10% polyacrylamide) under reducing (R) or non-reducing (N) conditions. The open arrow (Δ) indicates the position of TCRδ under reducing conditions and the continuous arrow (▶) indicates the position of delta TCR under non-reducing conditions. The size marker, M _r (in cloth), is shown on the left.

A) Radiolabeled cells in non-disulfide-linked γTCR (40KD) columns 1-6 to PBL-L2 were dissolved in 0.3% CHAPS detergent maintaining TCR-CD3 linkage, while immunoprecipitation in rows 7 and 8 It was carried out after chain separation (see method).

B) Radiolabeled cells in non-disulfide-linked γTCR (55KD) columns 1-4 to IDPS2 cells were lysed in 0.3% CHAPS detergent, while in the 5th and 6th rows, sedimentation was performed after chain separation.

C) WM-14 Cells Non-disulfide-linked γTCR (40KD) All rows correspond to immunoprecipitation in 1% Digitonin lysed radiolabeled cells.

D) Lysulfated non-disulfide-linked γTCR (40KD) radiolabeled thymic clone II cells in 1% Digitonin (rows 1-4) or 0.1% Triton X-100 (rows 5 and 6) On the other hand, immunoprecipitation was performed after chain separation in columns 7 and 8.

E) Non-disulfide-linked γTCR (40KD) to MOLT-13 leukemia T cells after dissolution in 0-3% CHAPS detergent of immunoprecipitation in columns 1-4, whereas immunoprecipitation in rows 5 and 6 After chain separation.

12 shows immunoprecipitation of γ TCR and δ TCR chains by anti-Cγml and anti-TCR δ 1 antibodies, respectively. MOLT-13 cells with radiolabeled cell surface were lysed in 0.3% CHAPS detergent and the γ, δ TCR-CD3 complex was isolated with an antibody of anti CD-3 monoclonal. Immunoprecipitates were analyzed by 10% SDS-PAGE under reducing conditions.

Row 1: immunoprecipitation with anti-Leu4 (anti-CD3) mAb.

Column 3: Immunoprecipitation by Anti-Cγ Antigen (anti-TCRΥ) mAb after Separation of Isolated γ, δ TCR-CD3 Complexes.

Row 4: Immunoprecipitation by Anti-TCR δ1 (Anti-TCRδ) mAb after Separation of Isolated γ, δ TCR-3CD3 Complexes.

Figure 13 Measurement of peptide backbone size and glycosylation of γ and δ TCRs from PEER and MOLT-13 cells. Monoclonal antibodies used for immunoprecipitation are anti-Cγml (anti-TCRγ), anti-TCRδ1 (anti-TCRδ) and P3 (labeled criteria) as shown at the top of each column. The leveled cell cultures used are indicated either at 10% SDS-PAGE radiographs or at the bottom of the pullograph. All samples are dissolved under reducing conditions. Size indication, M _r is in thousands.

A) a peptide backbone of γTCR from PEER and MOLT-13 cells, the size, the cells were labeled for 15 minutes with ³⁶ S- methionine biosynthesis formula S- cis and ³⁶ others. Samples were either endo H (+) or simulated (-) treated. Immunoprecipitation in anti-Cγml indicates the location of immature γTCRs in PEER cells (column 3) and MLOT-13 cells (column 7) while the corresponding polypeptide backbone size is visualized after treatment with endo H (columns 4 and 8). do.

B) Glycosylation of TCR from MOLT-13 Cells. ¹²⁵ I-labeled cells were immunoprecipitated with anti-CD-3mAb and incubated with δTCR polypeptide N-glycanase (column 4), endo H (column 2) or simulated (columns 1 and 3). Purification (see method).

14 shows the sequence of nucleotide sequence of MOLT-13 γTCR (form 2bc). Part A: Sequencing strategy of clone M13K. Partial restriction explanatory diagram of 1.1 kb cDNA clone M13K is shown. Part B: Nucleotide and inferred amino acid sequence of clone M13K. Signal sequence sequence (S), variable (V), N- | region (N), linkage (J) and constant (CI, CIIb, CIIc, CIII) region The gene sequence is indicated by an arrow and then It confirmed by comparing with the arrangement order. Lefranc et al., (1986 Cell 45: 237-246) (for S and V), Lefranc et al., (1986, Nature, 319: 420-422) and Quertermius et al., (1984, Immunol. 138 : 2687-2690) (for J) and Lefranx et al., (1986, Proc. Natl. Acal. Sci. USA 83: 9596-9600) and Pellicci et al., (1987, Science 237: 1051-1055) (About C). The deduced amino acid sequence starting from the initiator methionine is shown below the nucleotide sequence. Extracellular cysteine is indicated by a square. And potential N-linked carbohydrate attachment sites (N-X-S or N-X-T; Marshall, 1977, Ann. Rev. Biochem. 41: 673-702) are shown in parentheses.

15 is a preferred use of γ, δ TCR Form 1. Newly isolated terminal blood mononuclear cells from three healthy recipients were 125 I-labeled and lysed in 1% Triton X-100. Immunoprecipitates by P3 (technology, column 1 and 3) were analyzed under non-reducing conditions (N) and reducing conditions (R). The M _r marker in thousands is shown on the left.

FIG. 16 is a schematic of the three γ, δ TCR forms of men. CII exon encoded linker peptides are represented by squares (□) filled like Cγ 1 CII exon encoded peptides. ■, ■, ■ represent polypeptides encoded by Cγ2CII exon transcription a, transcription b and transcription c, respectively). Potential N-linked glycan attachment sites (O) and sulfhydryl groups (-SH) and imagined disulfide bridges (-S-S-).

Figure 17 is an explanatory diagram of the rearranged δTCR gene. Explanatory diagrams of γ119δ1 including EcoRI (RI), Hinc II (Hc), Scal (S) and PveII (P) positions and probes used in the blot analysis are shown.

Figure 18A is a schematic of the γTCR chain used for transfection with MOLT-13 cell culture water. This schematic is based on the reported analysis (Brenner, MB, 1986, Nature 322: 145-149, Brenner, M, B., et al., 1987, Nature 325: 689-694; Krangel, MS, et al ., 1987, Sience 237: 64-67). Pred., Represents predicted and 0bs., Represents observed. The size of the predicted glycosylated polypeptide uses all possible N-linked glycosylation sites (indicated by candy at the end of the bar), each containing 3KD of attached carbohydrates, and significant positional differences are introduced into other post-translational formulas. Measure The internal-chain disulfide bond forms of Ig-like molecules are also indicated. Cysteine residues (cys) are encoded as CII exons in the PBL C1γTCR, but it should be noted that these cysteines are not present in all transcriptions of the CII exons used in two different γTCR chains. In addition, 55 KD γTCR protein (Brenner, MB, et al., 1987, Nature 325: 689-694; Weiss, A. et al., 1986, Proc. Natl. Acad. Sci. USA 83: 6998-7002) was identified as IDP2. It is like that.

FIG. 18B used the expression plasmid constructs pFneo.PBL C1γ and pFneo.IDP2γ to inject γTCR clones into MOLT-13 cell cultures. PBL CLγTCRcDNA clones (PBL C1.15) and repaired IDP2γTCR cDNA clones (IDP2.11γ) (Krangel, MS, et al., 1987, Science 237: 64-67) were made to their parent plasmids by EcoRI digestion and CDNA was then ligated to SalI-cleaved and Klenow-treated pFneo mammalian expression delivery media. Clones containing cDNA inserts at appropriate locations relative to the virus forming the splenic foci (SFFV) were selected from the restriction diagram. pFneo (Saito, T., et el., 1987, Nature 325: 125-130) is a derivative of pTβFneo obtained by digesting rat βTCR cDNA inserts with BamHI and then ligation with T4 DNA synthase ( Ohashi, P, et al., 1985, Nature 316: 606-609. As indicated, this delivery medium contains the bacterial neomycin resistance gene (neo ^r ) under the control of the SV40 promoter and thus is resistant to antibody G418 in mammalian receptor cells. Gives resistance to The limiting position in these parentheses was destroyed during construction.

19 is an immunopotential analysis of γ, δ TCR in Molt-13 transspectant. Surface ¹²⁵ I-labeled cells were lysed in 0.3% CHAPS detergent to preserve chain dissociation and mAb P3 (baseline), anti-leu-4 (anti-CD-3), anti-TCRδ1 (anti-δTCR) or Immunoprecipitated with anti-Ti-γA (anti-Vγ2) and lysed by SDS-PAGE in non-reducing (N) or reducing (R) conditions and then visualized by radiographic methods as described previously (Brennet, MB, et al., 1986, Nature 322: 145-149; Brenner, M, B., et al., 1987, Nature 325: 689-694-). Anti-Ti-γA mAb exhibits a form of responsiveness to different T cell clones consistent with the recognition of vγ2 fragments. M13.PBL C1γ: MOLT-13 cells transfected with PBL C1-induced γTCR cDNA; Clone # 10 was used for this analysis. Size indicator, "(Molecular weight) shall be Daltons per thousand. Open arrow, residual MOLT-13 γTCR chain; Continuous arrows, transacted (PAL C1- or IDP2-derived) γTCR cDNA; # Table shows MOLT-13δTCR chains in non-reducing conditions. In other words, the δ TCR chain undergoes a change in mobility and co-moves with the 40KD chain. However, δ TCR chains are clearly visualized as 40KD bands under reducing conditions when 40KD γTCR proteins are not simultaneously immunoprecipitated, as shown by anti-Vγ2 anti-immunoprecipitation of M13.IDP2γ (column 19, FIG. 8).

Figure 20 is a two-dimensional gel analysis of γTCR polypeptides of the transpacket. 2 × 10 ⁷ cells were labeled with a surface ¹²⁵ I and treated with neuraminidase (150 mg of 1.5 mg PBS at 1.5 ° C. for 1.5 minutes) with 1 mg / ml of glucose and bovine serum albumin, respectively, and 0.3%. Dissolved in CHAPS detergent, immunoprecipitated with 1 μg anti-leu-4 mAb and analyzed 20 gels under reducing conditions. Non-equilibrium pH gradient gel electrophoresis (NEPHGE) was performed using Ampoline (LKB, Sweden) at pH 3.5 to 10 followed by SDS-polyacrylamide gel electrophoresis on a 10.5% acrylamide gel. (Brenner, MB, et al., 1987 Nature 325: 689-649). The location (not shown) of the CD3 component was used to identify and compare γTCR species expressed in different cell cultures. M13.PBL C1γ transfactant # 10 and M13.IDP2γ transfactant clone # 10 were used. Development arrow; MOLT-13γ TCR species; Continuous arrow, IDP2γTCR species; #Table, PBL C1γTC species.

21 degrees trans pack analysis of the polypeptide backbone in the chain size γTCR tanteu, the cells are shown as being ³⁶ S- methionine and S- cis and ³⁶ for 15 min pulse labeling with others P3 (reference), wherein -Cγml (wherein -γTCR ) Or anti-Ti-γA (anti-Vγ 2) mAb. Immunoprecipitation was either treated with endoglycosidase-H (Endo-H, +) or simulated-cultured (-), dissolved by SDS-PAGE and visualized by fluorescence. All samples were reduced. The M _r molecular weight label is in thousand daltons. Inclusion of an activity band serves as an additional internal marker of 43KD. M13.IDP2γ; MOLT-13 cells, M13.PBL C1γ transfected with ID2γ cDNA clone # 10; MOLT-13 cells transfected with PBL C1γTCR cDNA clone # 10.

22 is a blot analysis of γδ T cell clones and polyclonal human T cell populations. DNA from the genome was digested with EcoRI and tracked with a V-J probe. The source of DNA; PBL T-cell clones (columns 1,3-9), PBL (columns 10), neothymocytes (columns NBT-11), fetal thymic cells (columns FT-12) and B cells (germline-2 Lamellar-3Kd Vδ and 6.7 Kb Jδ fragments are shown on the left side of the blot, while five co-relocations of number IV are shown on the right, the size of the relocations from IV is 2.9 kb 3.5 kb 4.2 kb 6.2, respectively. kb and 7.1 kb.

5.1 γδ TCR Form 2bc Polypeptide and Nucleic Acid

The present invention relates to the form of a human γTCR polypeptide called 2bc (described in detail in sections 8 and 9 below). The invention also relates to nucleic acids encoding γ form 2bc, such as DNA and RNA, and these supplemental nucleic acids.

Form 1 and Form 2bc γTCR peptides are the previously reported forms of human γTCR (see PCT International Publication No. WO 88/00209, published January 4, 1988). Form 1 γTCR polypeptides have a molecular weight of about 40.000 daltons. Form 2bc γTCR polypeptides have a molecular weight of about 55,000 Daltons. Form 2bcγTCR chains have a slightly larger peptide backbone and comprise one excess potential N-linked glycan than Form 1. In contrast, the γTCR chain of the present invention in form 2bc has a molecular weight of about 40,000 Daltons. Moreover, Form 2bc γTCR polypeptides possess slightly smaller peptide backbones and 2-3 fewer N-linked glycans.

The γTCR chain form 2bc is more than 15KD (40 KD vs 55KD) or different in size compared to the form 2bc described above. This difference is calculated by reducing the amount of carbohydrate (5KD vs. 15KD) by 5KD smaller polypeptide backbone size (35KD vs. 40KD). Also approximately 35KD polypeptide backbone of Form 2bc distinguishes it from Form 1, which has a 40KD backbone size.

γTCR polypeptides differ from Form 1 and Form 2bc in contact with either Form 2bc or Region (Cγ) gene fragments. Form 1γTCR chain has a constant region encoded with a Cγ1 gene fragment containing a single CII exon (Krangel et al., 1987, Science 237: 64-67). Form 2abs γ polypeptides are encoded by a Cγ 2 gene fragment comprising three CII exons, namely transcription a, transcription b and transcription c (krangel et al., 1987 Science 237: 64-67; Littman et al., 1987 , Nature 326: 85-88). In contrast, one transcription of the sequence encoded by the Cγ2 second exon present in cDNA of form 2bc is lacking. Form 2bc contains two Cγ2 CII second exons, transcript b and transcription c. Transcription of CII, which is not present in Form 2bc, encodes a portion of the linkage region between the membrane and the extracellular domain.

Six potential N-linked carbohydrate attachment sites are present on the Form 2bc polypeptide. Biochemical data suggest that only 2-3 N-linked glycan membranes are attached to the polypeptide chain and thus do not use all potential positions.

In a specific embodiment the γTCR polypeptide form 2bc is MOLT-13 (Loh et al., 1987, Nature 330: 569-572) T cell culture or thymus induced clone II (Bank et al., 1986, Nature 322: 197-). 181). γTCR chain form 2bc can also be obtained from T lymphocytes of human subjects expressing this γTCR form.

Form 2bc γTCR polypeptides include the first amino acid sequence of the γTCR polypeptide shown in FIG. 14 or a portion thereof including a region consisting of transcription b and transcription c of Cγ2 CII.

The present invention also provides nucleic acid molecules encoding a γTCR type 2bc polypeptide having a molecular weight of about 40,000 Daltons. Only two of the three Cγ2 CII exons are obtained by translation of the nucleic acid sequence. The invention also relates to a nucleic acid sequence comprising a Cγ2 constant region with only two CII exons. This nucleic acid can be DNA, cDNA, RNA and supplemental nucleic acids and derivatives thereof. In a specific embodiment of the invention the DNA molecule comprises at least a portion of the nucleic acid sequence shown in FIG.

In the example discussed in paragraph 8, its encoding with a 2bc γTCR polypeptide is described. In the example described in section 9, the ability of the γTCR polypeptide to disulfide bond or glycosylate is measured in its primary region of the primary sequence.

5.2 Polypeptide Complexes Containing γTCR Form 2bc

The present invention relates to a polypeptide complex comprising γTCR chain form 2bc. In a specific embodiment the polypeptide complex consists of a T cell antigen receptor dimer. In particular such dimers include but are not limited to heterodimers (γ, δ heterodimers, γ, β heterodimers and α, γ heterodimers or γ, γ 'heterodimers in the form of 2bc or 2bc Or a homodimer.

In a particular embodiment of the invention the polypeptide complex comprising γTCR form 2bc is a γδTCR heterodimer. Such δTCR polypeptides comprise purified compounds comprising at least a portion of a γTCR form 2bc polypeptide provided by the present invention.

The polypeptide may have at least one inner chain, covalently disulfide bridge. This polypeptide also includes a δTCR polypeptide having a molecular weight of about 40,000 Daltons.

As shown in Example 9 below, the use of γTCR constant region CII exons (and such a primary sequence of γTCR chains) is characterized by differences in the size of cell surface γTCR proteins as well as the absence of disulfide bonds between TCR γ and δ. The amount of carbohydrate attached to γTCR, which is highly dependent on, is also measured. As such, the present invention also provides a method for making expression of a γTCR heterodimer of defined intramolecular bonds (disulfide or non-disulfide-bonded) and a particular γ into a cell expressing a δTCR chain and capable of expressing this γ gene. It provides a degree of γTCR glycosylation, including the insertion of a γTCR gene encoding a polypeptide form.

The present invention also provides a purified complex comprising a γTCR form 2bc polypeptide of the invention associated with a γTCR polypeptide (such as Form, 1, 2abc, or 2bc). In one embodiment of the invention two γTCR polypeptides are related to each other through at least one internal chain, covalent bond, disulfide bond. In another embodiment of the present invention two γTCR polypeptides are non-covalently related to each other. In another embodiment of the invention the two γTCR polypeptides have the same constant region. In another embodiment of the invention the two γTCR polypeptides have different constant regions.

5.3 Monoclonal Antibodies Reacting with γTCR Polypeptides

Monoclonal antibodies (mAb) against epitopes of γ or δ cell antigen receptors can be prepared using techniques for producing antibody molecules by continuous cell culture in culture. These include crude hybridoma technology and more recent human B cell hybridoma technology (Kozbor et al., 1983, Immunology Today 4: 72) as described by Kohler and Milstein (1975, Nature 256: 495-497). And EBV-Hybridoma technology (Cole et al., 1985, Monoclnal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

In one embodiment the monoclonal antibody may be a human monoclonal antibody or a chimeric human-mouse (or other species) monoclonal antibody. Antibodies of human monoclonal may be made by any of a number of techniques known in the art (eg, Teng et al., 1983, Proc Natl. Acad. Sci. USA 80: 7308-7312; Kozbor et al., 1983). , Immunolgy Today 4: 72-79; Olsson et al., 1982, Meth.Enzymol. 92: 3-16). Chimeric antibody molecules can also be prepared that include a consensus-binding region of a mouse (or rat or other species) having a certain region of humanity (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81). : 6851; Takeda et al., 1985, Nature 314: 452).

The present invention also relates to a method for identifying monoclonal antibodies that react with T cell antigen receptors. Such mAbs can be identified by detecting their ability to co-regulate CD3 antigens when binding mAbs to cells expressing both T cell antigen receptors and CD3 complexes. For example, CD3 co-regulation can be detected by measuring the amount of labeled anti-CD3 antibody bound to these cells. This method is described, for example, by the method of the following example 7.1.1 used to identify hybridoma secretory anti-V δ mAb δ TCAR-3.

Molecular clones of antibodies of epitopes of one γ or δ TCR polypeptide can be prepared by known techniques. Synthetic DNA methodology (see, eg, Maniatis et al., 1982, Molecular Cloning, A boratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) is used to encode monoclonal antibody molecules or their antibody binding regions. It can also be used to construct the nucleic acid sequence.

Antibody molecules may also be purified by known techniques such as, for example, immunoabsorption or immunoaffinity chromatography, chromatographic methods such as hHP (high performance liquid chromatography), or combinations thereof.

Antibody fragments comprising genotype molecules can be generated by known techniques. For example, such fragments include, but are not limited to: F (ab ') ₂ fragments that can be made by pepsin digestion of antibody molecules, Fab' fragments that can be generated by reducing disulfide bridges of F (ab ') ₂ fragments, and by reducing antibody molecules with papain and a reducing agent Fab fragments that can be made.

One embodiment of the invention relates to monoclonal antibodies that react with the variable region of the δ TCR chain. Such antibodies are δTACR-3 (aka TCSδ1) (see paragraph 7 below) that recognizes epitopes expressed from specific δ gene rearrangements. As described in section 7.2 below, mAbδTCAR-3 can stimulate the proliferation of γ, δ ⁺ T lymphocytes. Monoclonal antibody δTACR-3 can stimulate an increase in the lack of cytoplasmic γ, δ ⁺ T lymphocytes.

In another embodiment the invention relates to antibodies capable of reacting with certain regions of a γ or δ TCR polypeptide. In a specific embodiment the present invention relates to mAb TCRδ1 which reacts with a constant region of the δTCR chain (see next paragraph 6). In another specific embodiment, the invention relates to anti-Cγml that reacts with certain regions of the γTCR chain (see Section 8.1.7 below).

6. Generation of monoclonal antibody anti-TCRδ1 that specifically reacts with a region of the TCR δ subunit

6.1 Experimental Method

6.1.1 Cytopulograph Analysis of T Cell Cultures with Anti-TCRδ1

Anti-TCRδ1 mAb that specifically reacts with the δTCR chain region was made as follows. 1 gm of PEER cells were lysed in 50 ml of 0.3% CHAPS (3- [3-colamidofil) dimethyl-ammonio] 1-propanecellonate) detergent and 1 μl of UCHTL (Beverlery, PC, and Callard, 1981, Eur. J. Immunol. 11: 329-334) ascites, 500 μl of mAb 187.1 culture supernatant and Staphylococcus aureus Cowan I strain (SACI). Four intraperitoneal injections were placed at 6 week intervals followed by final enrichment of γδ TCR (without CD3) isolated by selective elution of γδ TCR from anti-immunocomplex using 2% Triton X-100. Elution was performed by both intravenous and intraperitoneal administration of the eluted material, and 4 days after this reinforcement, the mice were killed and melted as described previously (Brenner, M, B., et al., 1987, J. Immunol). 138: 1502-1509).

γδ TCR Cell Cultures PEER and IDP2 or αβTCR Cell Cultures HPB-MLT and JURKAT were stained with 50 μl of anti-TCRδ1 culture supernatant followed by an ortho-cydofulurograph analyte (see Figure 1). Stained with FITC-conjugated goat anti-mouse IgF (ab) ' ₂ fragment. Criteria were mAbs secreted with P3X63.Ag8 hybridoma (P3) and anti-CD3 mAbs were anti-Leu4 (Ledbetter, JA, et al., 1981, J. Exp. Med. 153: 310).

6.1.2 Immunochemical Analysis of Specificity of mAb Anti-TCRδ

Surface ¹²⁵ I-labeled IDP2 cells were lysed and their proteins were immunoprecipitated using reference mAb P3, anti-Led 4, anti-TCRδ1 or anti-Cγ serum. Precipitated samples were analyzed by SDS-PAGE and then radiographically. Γδ TCR and CD3 remained related in CHAPS detergent and immunoprecipitated as a complex by anti-Leu 4 (columns 2 and 3). However, after dissolving in 2% Triton X-100 detergent, anti-TCRδ immunoprecipitated γδTCR as a dimeric complex (column 6) without CD3 and anti-Leu 4 immunized CD3 as a trimeric complex without γδTCR (column 5). Precipitated. After separation of the γδ TCR-CD3 component, anti-TCRδ immunoprecipitated only TCRδ while anti-Cγ serum immunoprecipitated only TCRγ (column 9). Concerning the chain separation test (columns 7-9). Immunoprecipitated anti-Leu4 from CHAP lysed IDP2 cells were boiled in 1% SDS and then diluted to 4 volumes of 2% Triton X-100 and immunoprecipitated with anti-TCRδ1 or anti-Cγ serum. This follows the method described previously (Brenner, MB, et al., 1986, Nature 322: 145).

6.1.3 N-linked Glycosylation of TCRδ Polypeptides

¹²⁵ I-labeled IDP2 cells were lysed in 0.3% CHAPS, immunoprecipitated with anti-Leu4 and lysed by SDS-PAGE (see Figure 3). Reference sequence is a mock-digested IDP2δTCR polypeptide. N-glycanase digestion of δ TCR polypeptide was performed as follows. δ TCR was eluted with a gel thin film and then eluted with N-glycanase (Genzme Corp.) and digested (10 U / ml) with 30 μl of 0.17% SDS, 1.25% Nonidet P-40, 0.2M Natarium Phosphate Buffer pH 8.6 It was carried out at 37 ℃ for 16 hours. Digested or mock-cultured δ TCR samples were analyzed by SDS-PAGE and visualized by radiographic methods.

6.1.4 Recognition of in vitro translation products of cDNA clone IDP20-240 / 38 by mAb anti-TCR δ1

The plasmid designating pGEM3-0-240 / 38 was constructed as follows and used for in vitro transcription-translation (FIG. 4). Insertion of IDP2 0-240 / 38 (δTCR) cDNA clone 1.5 kB begins in the codeine of composition group D sequence and includes the rest of the coding region and mostly the 3 ′ beaver region. This insertion was split as a single EcoRI fragment from the λgt10 arm by partial EcoRI digestion (to prevent splitting of the internal EcoRI site). This fragment was subcloned into a blueclip delivery medium (Stratagene.) The following insert inserts a single BamH-SalI fragment (A) to facilitate directional cloning into pGEM- (Promega Biltech) downstream of the T7 promoter. The ends were removed from the delivery medium as from BlueScript delivery media multiplexers.Linearized with the resulting pGEM3-0-240 / 38 plasmid SamI and capped into synthetic transcripts using T7 RNA polymerase (Kranger MS, et al., 1987, Science 237: 64) Completion and size of transcripts were followed by aliquots of reaction mixtures comprising P-ATP Single RNA species of 1.5 Kb was observed in vitro in the presence of S-methionine My translation was performed on rabbit reticulocyte degradant After in vitro translation the sample was boiled in 2 mM dithiothreitolide 1% SDS and then 2% in Tris buffered saline pH7.5 Ten doses of Liton X-100 were added, samples were immunoprecipitated with reference mAb P3 (columns 1 and 3 of FIG. 5) or anti-TRCδ1 mAb (columns 2 and 4) and then with SDS-PAGE and then fluoro. Analyzed by chromatography (Bonner WJ, and Laskey, RA, 1974, Eur. J. Biochem. 46: 83-88).

6.2 Experiment Result

We generated a monoclonal antibody (mAb), anti-TCR δ1 that specifically reacts with a region of δ TCR.

Γδ TCR-CD3 complex from PEER cell culture (Weiss, A., et al., 1986, Proc. Natl. Acad. Sci. USA 83: 6998-7002; Brenne, MB, et al., 1987, Nature 325: 689-694) was used as an immunogen in the production of antibody-secreting hardoma cells. Hybridomas secreted PEER protein by cell surface binding (cytopuluographic analysis) and immunoprecipitation, followed by SDS-PAGE analysis. Two hybridoma supernatants (5A6 and 4A1) bound to the surface of PEER cells. After subcloning one mAb (5A6.E9) was characterized again. This mAb bound to the surface of γδ TCR lymphocytes (PEER, IDP2) but failed the response of αβTCR cells (HPB-MLT, JURKAT) or non-T lymphocytes (Figures 1 and data not shown). Although the immunogen consists of a complex of γδ TCR and CD3, the greater affinity of the mAb to the presented mAb in γδ TCR cell cultures was not intended to counter CD3 determinants.

The specificity of this mAb was measured in immunoprecipitation studies using various determinants that influence the involvement of proteins including receptor complexes. ¹²⁵ I-leveled IDP2 cells were lysed in CHAPS determinants, but TCRγ and δ and CD3 γ, δ and subunits remained part of the associated complexes immunoprecipitated by anti-CD3 antibodies (FIGS. 3 and 4) ). However, if radiation-labeled IDP2 cells were lysed in 2% Triton X-100 detergent, γδ TCR and CD3 were greatly dissociated and selective precipitation of CD3 was achieved with the use of anti-CD3 mAb (Fig. 5, column 5). Under these latter conditions, mAb 5A6.E9 immunoprecipitated γδ TCR as a heterodimer without associated CD3 (column FIG. 6). This observer presents the first indication as evidence that TCRγ and TCRδ are present as non-disulfide-bonded heterodimers. In order to determine whether mAb 5A6.E9 reacted with γTCR chain, δTCR chain or binding determinant, immunoprecipitation of the isolated polypeptide chain was performed. Anti-Leu4 immunocompetants from CHAPS-lysed IDP2 cells with radiation labeling were boiled in 1% SDS to dissociate γTCR, δTCR and CD3 proteins. After 4 volumes of 2% Triton X-100 dissociation, mAb 5A6.E9 specifically immunoprecipitated 40KD (δTCR) species (Figure 3 column 7). When the same immunoprecipitates were analyzed under reduced conditions (column 2, column 8), a dramatic change in SDS-PAGE mobility was observed. This phenomenon is characteristic of δ TCRs from IDP2 and PEET cell cultures (Brenner, MB, et al., 1987, Nature 325-694). In contrast, when the separated chains were immunoprecipitated with anti-Cγ serum, 55KD species (γTCR) were immunoprecipitated instead of 40KD species (δTCR) (FIG. 9, column 9). Based on these biochemical and surface binding studies, mAb 5A6.E9 becomes anti-TCRδ1.

In addition to PEER and IDP2, anti-TCRδ1 also immunoprecipitated TCRδ from other γδ TCR cell cultures including MOLT-13 and PBL culture 2. Furthermore, experiments show that anti-TCRδ1 reacts with the determinants encoded by this TCRδ constant (C) gene fragment.

We isolated cDNA clones from IDP2 cell cultures (eg IDP20-240 / 38) by a subtraction solution representing the gene encoding the TCRδ subunit. In blot experiments, genes hybridized by IDP2 group 0 cDNA clones were expressed and rearranged in γδ TCR lymphocytes, but not representatively (and often eliminated) in αβTCR cells, compared to other cDNA clones in sequence comparison with other TCR genes. Is shown to consist of novel V, D (?), J and C gene fragments. The IDP2 Group I composition DNA sequence includes a long, open dominant frame representing a polypeptide having two potential asparagine-linked glycosylation sites and a molecular weight of 31.3 kilodalto. To determine the molecular weight of the aglycosylated δTCR protein and the number of asparagine-linked carbohydrates present in the mature IDPS2 δTCR polypeptide, gel-purified δTCR was treated with N-glycanase or mock-cultured and analyzed by SDS-PAGE. (Article 3). Removing N-linked carbohydrates results in a 5KD reduction (40KD to 35KD) in apparent molecular weight, suggesting that there are two (2.5-3KD) N-linked glycans in IDP2δTCR. It is well correlated with the number of N-linked glycans represented by the translated amino acid sequence. The apparent molecular weight of the protein generally agrees differently with that shown at 3.7 KD.

Given the specificity of the anti-TCR δ1 reactive δTCR polypeptide in IDP2 cells and the phosphorylation of a partially denatured (sDS boiled) δTCR, I would like to know whether these mAbs directly recognize the polypeptide encoded by the δTCR cDNA clone. Tested. Thus inserts from cDNA clone IDP2 0-240 / 38 were western cloned into the pGEM-3 expression delivery media under the T7 promoter (4th). Next, T7 RNA polymerase was used in in vitro reticulocyte depletion of transcripts generated in vitro to direct the synthesis of the protein in the presence of ³⁶ S-methioin. Following in vitro transcription-translation the reaction mixture was diluted in 1% SDS, diluted to 10 volumes of 2% Triton X-100 and immunoprecipitated with streamlined-differential reference mAb or anti-TCRδ1. Anti-TCRδ ⁺ mAb specifically immunoprecipitated the major species (34KD) (FIG. 5, column 4). These bands were not observed in immunoprecipitates when using the reference mAb (3), excluding RNA transcripts (1 and 2), or when using the γTCR construct. This species with radiolabeled immunoprecipitated by mAb anti-TCRδ1 corresponds to the δ polypeptide whose synthesis is specifically directed by the IDP2 0-240 / 38 cDNA clone. This polypeptide (34KD) is very similar in size to the N-glycanized IDP2 δ TCR chain (35KD). The IDP2 0-240 / 38 clone lacks the native ATG initiation condon as well as the leader sequence. Within the V region of this codon are two potential internal ATG codons (residues 12 and 44) (Figure 5). Use of such codons to initiate the synthesis results in one or more polypeptide species that calculate possible or small amounts of the species indicated (FIG. 4, column 4). Thus, direct serological recognition is achieved by anti-TCRδ1 of the IDP2 δTCR subunit encoded by clones IDP2 0-240 / 38.

7. Generation of Monoclonal Antibody δTACR-3 Reacting Specificly with the TCRdelta Subunit Variable Region

7.1 Experimental Method

7.1.1 Immunoprecipitation and SDS-PAGE Analysis of T Cell Antigen Receptors

The δTCR-3 mAb, which specifically reacts with the variable region of the δTCR chain, was generated as follows. One rat was immunized with 2 × 10 ⁷ Molt-13 cells by intraperitoneal injection. One month later, mice were enriched with 1 × 10 ⁷ Molt-13 cells by intravenous injection daily for the next 3 days and then melted in the presence of 50% polyethylene glycol 1500 with P × 63Ag8.653 cells as immune splenocytes. Hybridomas were filled by analyzing CD3 co-regulation with a flow cytometer. Analysis of CD3 co-regulation was based on the observation that antibodies to α, β T cell antigen receptors result in the incorporation of CD3 complexes when cultured into cells (Lainer, L, L., et al., 1986, J. Immunol. 137: 2286; Meuer, SC, et al., 1983, J. Exp. Med. 157: 705).

Molt-13, PEER and HPB-all cell lines were iodinated using lactoperoxidase technology. ¹²⁵ I-βF1 labeled cells were lysed in Tris-buffered saline (pH 8) containing 1% Triton X-100. The pulverized product was immunoprecipitated using the δTCR-3 antibody or βF1 antibody. βF1 is a skeletal monoclonal antibody against the TCR chain (Brenner, MB, et al., 1987, J. Immunol. 138: 1509). All samples were analyzed by SDS-PAGE in reducing or non-reducing conditions (Figure 6). The two Molt-13 and PEER all CD3 ⁺ 4 ^- 8 ^- WT31 ^- a. HPB is CD3 ⁺ 4 ^- the WT31 ^{+ -} 8.

As shown in FIG. 6, δ TCR immunoprecipitated non-disulfide-linked γ and δ chains from Molt-13 and PEER cells, while βF1 disulfide-linked α and from all HPE-cells. β chains were immunoprecipitated. The difference in radiographic intensity between the bands corresponding to the δ and γ chains indicates a difference in the degree of iodide these two proteins.

7.1.2 Immunoprecipitation of δTCR Chains by δTCR-3 Antibodies

FIG. 7 was dissolved in Tris-buffered saline (pH 8) or 0.3% Triton X-100 containing 0.3% CHAPS (3- [3-colamidopropyl) dimethylammonium] 1-flopansulfonate). In 1% Triton X-100 γδ TCR dissociates from the CD3 complex, while in 0.3% CHAPS γδTCR remains associated with the CD3 complex. I-labeled digests used in lines 3, 4, and 7 of the immunoprecipitation were denatured by adding SDS to a final concentration of 1% and heating to 68 ° C. for 5 minutes. After cooling iodoacetoamide was added to a final concentration of 20 mM. This mixture was then diluted with 4 volumes of 1.5% Triton X-100 in Tris-buffered saline (pH 8). This denaturation completely dissociates γ chain, δ chain and CD3 proteins from one another. All samples were analyzed by SDS-PAGE under non-reducing conditions (N) except for four rows of samples under non-reducing conditions (R) except for four rows of samples under reducing conditions (R). Note the difference in the mobility of the δ chain in reducing and non-reducing conditions. Anti-Cγ antigens were generated by immunizing rabbits with 20 amino acid synthetic peptides from the γ constant region (residues 117-136).

7.1.3. Analysis of cell surface staining by flow cytometry

5 × 10 5 cells were incubated at 4 ° C. for 30 min with appropriate antibody (NMS (serum of normal rats), δTCR-3, OKT3 or WT31). After washing the cells were incubated for 30 minutes at 4 ° C. with phycoerythrin (PE) -conjugated goat anti-mouse IgG. After washing the cells were incubated for 30 min at 4 ° C. with fluorescence (FITC) -conjugated IKT3 and the cells were then graphically analyzed with ortho cytopuluo (FIG. 9).

7.1.5. Determination of Inclusion Body Ca2 ⁺ Concentration ([Ca2 ⁺ ] ₁ ) vs. Time in Cytoplasm

Molt-13 cells were prepared in the form of aceoxymethyl ester, Ca2 ⁺ -sensitive probe fura-2 (2 μm from 1 mM stock of dimethyl sulfoxide, Molecular Probes, Eugene, Oregon) at 10 ⁷ cells / ml and 10% at RPMI 1640. Labeling was performed at 37 ° C. for 30 minutes at the concentration of fetal bovine serum. The cells were then washed and resuspended in 10 ⁷ cells / ml and 5% fetal bovine serum in Hanks balanced saline solution (HBSS) and stored dark at room temperature until use. Fluorescence 2 × 10 ⁶ cells were centrifuged and then resuspended in 2 ml of fresh HBSS and placed at 37 ° C. in a quartz cuvette and stirred constantly. Fluorescence was measured in cell suspension on an SPR-500C fluorometer (SLM Aminoco, Urbana, Iiilinois) and excitation wavelengths varying between -340 (± 2) and 380 (±) nm and radiation were detected at 510 (± 5) nm. . The ratio of 350/380 was automatically calculated (one ratio every two seconds), constructed and stored at IBM PC AT. Quantification of [Ca2 ⁺ ] ₁ from fluorescence ratio was performed as described by Grynkiewcz et al (1985, J. Biol Chem. 260: 3440). Addition of extraneous antibodies did not change [Ca2 ⁺ ] ₁ , whereas cytolysis resulted in 1 μm of [Ca2 ⁺ ] ₁ .

7.2 Results

We have generated a monoclonal antibody δTCR-3 that can be used to characterize δ polypeptides and to face different regions of the TCR δ chain. This monoclonal antibody binds to T cells with γδ TCR and also induces a fura-2Ca2 ⁺ signal when bound to Molt-13 cells.

δTCR-3 antibody is a monoclonal CD3 ⁺ 4 ^- was generated by immunizing a rat cell cultures mainly ^- 8 ^- Molt-13 having the WT31 ⁺ phenotypes. Hybridomas were filled with CD-co-regulation at the run. Wild clones were further filled with immunoprecipitation. Δ TCR-3 immunoprecipitation of γδ TCR heterodimer from ¹²⁵ I-labeled Molt-13 and PEER degradation products is shown in FIG. 6. δ TCR-3 does not immunoprecipitate any polypeptide from HPB-ALL (FIG. 6). Conversely, βF1 (Brenner, MB, et al., 1987, J. Immunol. 138: 1502-1509), a monoclonal antibody with skeletal specificity in β chains, is a diclonal αβ heterolog from HPB-all cell cultures. The body is immunoprecipitated (Figures 6, 10 and 12). When analyzed under reducing or non-reducing conditions, immunoprecipitated γδ receptors from both Molt-13 and cells show a heteroterodimeric structure showing non-disulfide-linked γδ TCR in these two cell cultures. There is a slight change in the mobility of the δ chains in reducing conditions compared to those observed in non-reducing conditions (Figures 6, 1 and 3 and 5 and 7), which is a noted phenomenon in IDP2 and PEER cell cultures ( Brenner, MB, et al., 1987, Nature 325: 694), suggesting the presence of disulfide bonds in the inner chain. Immunoprecipitation was performed using ¹²⁵ I-labeled Molt-13 cytolysis products dissolved in 0.3% CHAPS detergent to indicate that the δTCR-3 antibody recognizes CD3-related γTCR (column 1, FIG. 1). . Under these conditions, the CD complex remained associated with the receptor and immunoprecipitated both γδ heterodimer and CD3 complex with δTCR-3. However, only the γδ hyterodimer of the ¹²⁵ I-labeled digest was immunoprecipitated with δTCR-3 (FIG. 7, column 2). As a reference, UCHT-1 (Beverley, PC, and Callard RE, 1981, Eur. J. Immunol. 11: 329-334), an anti-CD3 antibody, immunoprecipitates only CD3 complexes but does not immunoprecipitate γδ heterodimers ( 7, column 5).

The specificity of δTCR-3 was further analyzed using immunoprecipitates of the modified ¹²⁵ I-labeled Molt-13 digest with γδTCR and CD3-protein completely dissociated. δ TCR-3 specifically immunoprecipitated δ chains having a clear molecular weight of 38 KD (FIG. 7, column 3) under non-reducing conditions and a molecular weight of 40 KD (column 7 FIG. 4) under reducing conditions. Γ chains with anti-Cγ antiserum were immunoprecipitated (FIG. 7, column 7). These data indicate that δ TCR-3 is a δ chain.

δTCR-3 not only immunoprecipitates γδTCR heterodimers from PEER and Molt-13 cell cultures but also binds to the surface of these cell cultures and to IDP2 clones (Brenner, M., et al., 1987, Nature 325: 689-694). It does not bind to HPB-ALL and Jurkat cell cultures with αβTCR- (Figure 8). Conversely, WT31 (Tax, WJM, et al., 1983 Naure 304: 445-447), a monoclonal antibody to skeletal antibodies to αβTCR, responds to αβTCR-positive HPB-ALL and Jurkat cell cultures, but does It does not react with PEER and IDP2 cells (Figure 8). When testing normal peripheral blood lymphocytes (PBLs), the depleted population of CD3 ⁺ lymphocytes (0.9-2.4%) was positive for δ TCR-3 (Figure 9). When used for culturing normal human PBL to δTCR-3 immobilized on a tissue culture plate, it selectively stimulated the proliferation of γδ TCR-positive wasted population. After 45 days in culture, the γδ TCR depleted group showed 96% of the total cell count.

Antibodies to [alpha] [beta] T cell antigen receptors stimulate elevation of cell morphology free calcium ion concentration [Ca ²⁺ ] ₁ (Weiss A., et al., 1986 m, ann. Rev. Immunol. 4: 593). Incubation of Molt-13 cells with δ TCR-3 caused a sharp increase in [Ca ²⁺ ] ₁ , similar to the response induced with anti-T3 antibodies (FIG. 10). Moreover, δTCR-3 similarly stimulated Ca2 + flux in PEER cells and γδ TCR-positive cell cultures generated from PBL as described above. We also observed that culture of Molt-13, PEER and IDP2 cells by δTCAR-3 caused co-regulation of CD3 protein complexes.

The epitope specificity of mAb δTCR-3 (also known as mAb TCRδ1) is described in 11.2.2. It is shown in a term.

8. Three Forms of Human T Cell Receptor γδ: Selective Use of One Form of Selected Healthy Individuals

Herein is shown the structure of a new form of human T cell receptor γδ (γδ TCR) consisting of 40KD TCRγ glycoproteins associated non-covalently by TCRδ chains. The newly identified γTCR glycoprotein, called form 2bc, differs in size by more than 15KD (40KD vs 55KD) compared to the non-disulfide-bound TCRγ form (form 2abc) described above. This difference is calculated by the 5KD smaller polypeptide backbone size (35KD vs. 40KD) and by the reduction in the amount of carbohydrates (5KD vs. 15KD). Two-nucleotide sequence analysis of cDNA clones corresponding to Form 2bc revealed that transcription of a constant region (Cγ2) second exon in which the Form 2bc cDNA clone is present in the cDNA of another nondisulfide-linked TCRγ subunit (Form 2abc) Indicates that it is lacking. This CII exon transcription encodes the portion of the linker region between the transmembrane region of the membrane and a region of the cell. Since the potential N-linked carbohydrate attachment sites are the same in both non-disulfide-bonded forms, the linker region that we conclude may affect the formation of proteins, thus affecting the amount of carbohydrates attached. Will be. In contrast, the δTCR subunit of this γδ TCR type shows no change in the peptide backbone size and peptide explanatory analysis.

We also tested the use of three forms of γδ TCR complexes in peripheral blood. Almost independent use of form 1 disulfide-bound form was observed in some healthy subjects. In some days Form 1 expressed with Form 2bc. Form 2abc was not identified in the subjects tested.

8.1.2. Experiment method

8.1.1 Antibodies

Monoclonal antibodies used were anti-Leu 4 (anti-CD3) (Ledbetter, et al., 1981, J. Exp Med. 153: 310-323). ΒF1 (anti-βTCR) (Brenner, et al., 1987 , J. Immunol. 138: 1502-1509), anti-TCRδ 1 (anti-δTCR) (reacts with the δTCR chain scheduling region described in paragraph 6 above), P3 (reference) (P3X63.Ag8; secreted by , Koehlet and Milsteim, 1975, Nature 256: 495-497), 187. 1 (anti-mouse light chain of rats) (Yelton et al., 1981, Hybridoma 1: 5-11), and WT31 (αβTCR lymphocytes brighten) (Spits et al., 1985, J. Immunol. 135: 1922-1928.) Anti Cγb peptido serum (anti-γTCR) was converted to a 22 amino acid synthetic peptide (Gln-Leu-Asp-Val-Ser-Pro-Lys). -Pro-Lys-Pro-Thr0Ile-Phe-Leu-Pro-Ser-Ila-Ala-Glu-Thr-Lys-Cys) (PCT Iternational Publication NO.Wo 88/00209, published January 14, 1988) .

8.1.2. Cell culture

PEER (Weiss, et al., 1986, Natl. Acad. Sci. USA 83: 6998-7002 and MOLT-13iisolated by J. Minowada, Loh et al., 1987, Nature 330: 569-572) cultured T leukemia cells Lord. Clonal M-14 from umbilical cord blood (Alarcon et al., 1987, Proc. Natl. Acad. Sci. USA 84: 3861-3865) and cell culture IDP2 from peripheral blood (Brenner et al., 1986, Nature 322) : 145-149; PCT Iternational Publication NO.Wo 88/00209, published January 14, 1988) and thymus-induced clone II (Bank et al., 1986, Nature 322: 179-181) as described above. It was. Cell culture line 2 (PBL-L2) obtained from peripheral blood was isolated by sorting peripheral blood isolated lymphocytes that were not stained with mAb WT31. The isolated cells were then expanded in vitro into PRMI 1640 medium supplemented with 10% (V / V) conditioned medium containing IL0-2 and 10% (V / V) human serum as injector cells of the cock that were examined and examined. Stimulated every three weeks.

8.1.3. Iodide and Immunoprecipitation

2 × 10 ⁷ cells were isolated by Ficoll-diatrixoate (Organon Teknida Corp.) centrifugation and 5 mM glucose and 1 mM Ci (I England Nuclear) added with 1 mM MgCl, 100 μg of lactoperoxidase (80-100 U / mg, Sigma). Iodine on ice in 0.5 ml of phosphate buffer saline, pH 7.4 (PBS). 10 μl of 0.03% hydrogen peroxide solution was added for 30 minutes at 5 minute intervals during the reaction. Cells were dissolved overnight in detergent supplemented TBS (50 mM Tris-Base pH 7.6, 140 mM NaCl) containing 1 mM phenylmethylsulfonyl (PMAF sigma) and 8 mM iodine acetamide (TAA, Sigma). As shown, the different detergents used in this study were 0.3% (W / V) 3-[(3-Clamidopropyl) dimitylammonio] 1-propane-sulfonate (CHAPS, Sigma) 1% (W / V ) Digitonin (Aldrich) and Triton X-100 (TX-100, Sigma). After centrifugation at 100,000xg for 20 minutes to remove insoluble materials, detergent digests were incubated for 30 minutes with 4μl normal rabbit serum (NRS) and 400μl 187.1 hybridoma culture medium and then fixed with Staphylococcus aureus Cowan I ( It was pre-purified by adding 10% (WV) cell suspension of Staphylococcus aureus Cow I) (Pansorbin, Calbiochem). After 1 hour incubation, pansorbin was removed by centrifugation. Unusual precipitation added 0.25 μl βF1 ascites, 1 μl of 1 mg / ml anti-Leu 4 or 0.25 μl P3 ascendant to each sample, and 150 μl of 187.1 culture supernatant and the mixture was vibrated at 4 ° C. for 1 hour. Immunoprecipitates were washed five times with 0.1% (V / V) Triton X-100 containing TBS and analyzed by dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970, Nature 227: 690). -685).

Cells iodinated for immunoprecipitation with anti-Cγb peptide serum were lysed in 1% (W / V) sodium dodecyl sulfate (SDS) containing TBS and then boiled for 3 minutes. After cooling, a mixture of 1 mg / ml deoxyribonuclease (DNAse) and 0.5 mg / ml RNAse in 5% 50% MgCl in 2% (V / V) Troton X-100 in PBS containing PMSF and IAA Add with 200 μl. As described above, 187.1 mAb was excluded and prepurification and immunoprecipitation were performed. Immunoprecipitates were washed in TBS containing 0.5% (V / V) Triton X-100, 0.5% (W / V) SDS.

8.1.4. Biosynthetic Labeling

Cells grown at 4 × 10 exponential expression were resuspended in 4 ml of methionine cysteine-free RPMI 1640 (Select-Amine Kit, Gibco) supplemented with 10% dialysed fetal bovine serum (FCS) and 20 mM rounds. After a 30 min asa period at 37 ° C. 1 mCi of ³⁶ S-methionine 1 mCi of ³⁶ S-cysteine was added and allowed for 15 minutes of labeling. Cells were harvested and lysed in 2% (V / V) Triton X-100, TBS. Pre-purification and immunoprecipitation were performed as described above as described above. This immunoprecipitate was washed four times in 0.5% (V / V) Triton X-100, 0.5% (W / V) deoxycholic acid, 0.05% (W / V) SDS, TBS, followed by 0.5% (V / V). V) washed three times with Triton X-100, 0.5N NaCl, 5 mM EDTA, 50 mM Tris, pH 7.6. This sample was analyzed by SDS-PAGE and visualized by standard pluography methods (Bonner and Laskey, 1974, Eur. J. Biochem. 46; 83-88).

8.1.5. Gel Purification of δ TCR Protein

Surface iodinated cells were lysed in 0.3% (W / V) CHAPS-TBS and immunoprecipitated using 50 μl of anti-Leu4-bound Sepharose drops. Immunoprecipitated species were dissolved in SDS-PAGE under reducing conditions and the wet gel was exposed to XAR-5 film (Kodak) for 24 hours at 4 ° C. to visualize radiolabeled δTCR protein. The gel region corresponding to δ TCR was excised and incubated in 5% (V / V) 2-mercaptoethanol containing sample buffer and lysed twice by SDS-PAGE. Because of the specific SDS-PAGE mobility change upon reduction, the δTCR protein could be isolated and subsequently purified from contaminants. TCR protein was incubated overnight at 37 ° C. in 0.5% (W / V) SDS, 50 mM ammonium bicarbonate buffer and lyophilized.

8.1.6. Endoglycosidase Digestion

For the endoglycosidase H (Endo H) digestion, the immunoprecipitated material or gel purified protein was agitated for 30 minutes in 40 μl of 1% (W / V) SDS-solution containing 0.14M 2-mercaptoethanol. After cooling the mixture was diluted with 360 μl of 0.15 M acetate buffer pH 5.5 containing 1 mM PMSF. 5 μl of Endo H (1 U / ml-Endo-β-N-acetylr glycosamidase H, Genzyme) was incubated for 14 hours at 37 ° C. as half of the above solution and the other half was simulated.

Gel-purified material for N-glycanase (NOGLY) digestion was boiled for 3 min in 35 μl of 0.05% (W / V) SDS, 0.01 M 2-mercaptoethanol. Then 100 μl of 0.2 M sodium phosphate (pH 8.6), 1.25% (V / V) Triton X-100 was added. Half of this mixture was added 1 μl of N-glycanase (250 U / ml, peptide-N- [N-acetyl-β-glucoamiminyl] asparagine amidase (genzyme) was incubated at 37 ° C. for 16 hours while the other half was simulated.

After digestion, 10 μg of bovine serum albumin was added as a carrier and the sample was recovered by trichloroacetic acid precipitation. Protein pellets were taken in sample buffer containing 5% (V / V) 2-mercapto ethanol.

8.1.7. Production of Monoclonal Antibody, Anti-Cγml

The fraction of Cγ CI and CII exons of HPB-MLT pTγ- was isolated using the BamHI and PstI positions at nucleotides 571 and 848 (Dialynas et al., 1986, Proc. Nat l. Acak. Sci. USA 83 : 2619-2623) and cloned into the expression delivery medium pRIT2T (Pharmacia). The resulting Protein A molten protein was expressed in E. coli N4830. The bacteria were digested with lysozyme and the molten protein was isolated by purification on an IgG Sepharose column. Mice were injected intraperitoneally with 100 μg of lysed protein in Freund's adjuvant on days 0, 7, and 28. After 28 days, 100 μg of lysed protein was injected intravenously into PBS. After 3 days splenocytes were isolated and fused with hybridoma P3X63Ag8.653 as described (Brenner et al., 1987, J. Imunol. 138: 1502-1509). Hybridomas were filled with ELISA. 96 well bottom flat plates (LINBRO, Flow Laboratories) were incubated overnight in 0.4 μg of molten or non-fused protein in PBS. Nonspecific binding sites were blocked at 23 ° C. as 0.25 mg / ml normal rabbit IgG (Sigma) in PBS containing 50% (V / V) FCS. 50 μl of hybridoma supernatant was added at 4 ° C. for 1 hour and then incubated similarly in 50 μl of 5 μg / ml solution of peroxidase-conjugated anti-rat IgG (Cappel). All cultures Intersequence was performed with a washing step using 10% (V / V) FCS, 0.1% (W / V) BSA, PBS. ELISA was developed with 0.8% (W / V) O-phenylenediamine (Sigma) in 0.012% (W / V) hydrogen peroxide, containing phosphate-citrate buffer, pH 5.0.

Although anti-Cγml (IgG) does not recognize native γδTCR / CE3 complexes in cytopulologic analysis and also does not recognize γδTCR heterodimers from Triton X-100 lysed cells in immunoprecipitation Cγml (IgG) recognizes γTCR precursors and mature γTCR proteins labeled biosynthetically after CD3 / γδ TCR proteins are separated into individual chains. In this way, anti-Cγml was expressed by boiling anti-CD3 immunoprecipitates in TBS at 1% (W / V) SDS to separate the CD3 / γδTCR complex into individual chains and then phosphorylating the γTCR protein. 12 degrees, 3 rows).

8.1.83. Isolation and Sequence of MLOT-13 γTCR cDNA Clone

Poly (A) + RNA was prepared from MOLT-13 cells as urea / lithium chloride precipitate followed by oligo (dT) cellulose affinity chromatography. Method of Huynh et al., 1985 using Mung Bean Nuclease for λgt10 cDNA libraries in hairpin loop splitting (McMutcham et al., 1984, Science 225: 626-628) DNA Cloning, Glover, DM ed IRI Press, Oxford, I:. to 49-78) was prepared from poly (a) ⁺ RNA. This cDNA library was amplified in E. coil strain C600 Hlf and spot-filtered hybrid with PTγI (Dialynas et al., 1986, Proc. Natl. Acad. Sci. USA 83: 2619-2623) with ³² P-labels. It was filled with a cutting board. Positive clones were analyzed to determine size and restriction enzyme diagram and cDNA clone M13K was selected to know the sequence. CDNA of M13K was excised from λgt10 phage as endonuclease EcoRI and digested again with appropriate restriction enzymes. Dideoxy chain termination method (Sanger et al., 1977, Proc. Nal. Acad. Sci. USA 74) using a modified T7 polymerase (Sequenase, United States Biochemical Corp) subcloned into the M13 delivery medium. 5463-5467) to determine the arrangement order.

Clone M13K corresponds to full length γTCR transcription in a frame comprising 36 nucleotides of the 5 'untranslated region and 3 nucleotides 72 nucleotides (Figure 14). The nucleotide sequence of the V region was identical to that of the genome Vγ 1.3 sequence except for the C to T (Ile to Val) changes of nucleotide 53 in the imaginary signal sequence (nomenclature Lefrance et al., 1986a, cell 45: 237-246; Strauss et al., 1987, Science 237: 1217-1219). The J region is identical to the Jγ 2.3 sequence (nomenclature based of Lefrance et al., 1986b, Nature 319: 420-422; Quetermous et al., 1987, J. Ommunol. 138: 2687-2690). Interestingly, 8 nucleotides do not appear to be infused by the V or J sequence of the genome but rather occur at the V-J junctions representing the N-region. The C region sequence is identical to the corresponding genomic sequence except for nucleotides 559 (G to C; Val to Ile) and nucleotides 908 (T to C; Met to Thr). (Lfranc et al., 1986c, Proc. Nat l. Acad. Sci. USA 863: 9596-9600).

8.2. result

8.1.2. New γδ TCR Protein Complex

A preliminary study of peripheral blood γδ TCR lymphocytes indicates the presence of different CD3-related complexes from the human γδTCR form known. With the intention of describing this form, they have created and characterized a number of cell cultures from normal human donors. [Alpha] βTCR lymphocytes remaining in peripheral blood lymphocytes were stained as monoclonal antibody (mAb) WT31 which brightly stained. Unstained cells were isolated as cell sorting and then expanded in IL-2 with medium in vitro. Peripheral blood lymphocyte culture 2 (PBL-L2) obtained by this method was demonstrated to be homogeneous CD3 ⁺ CD4 ^- CD8 ^- cells of γδ TCR lymphocytes.

In order to visualize the γδ TCR complex in the cells on PBL-L2, immunoprecipitation was performed on cells with ¹²⁵ I-labeled cell surface dissolved in CHAPS or Digitonin. In this world, the physical association between the CD3 complex and the γδ TCR subunit is preserved. SDS-PAGE of anti-CD3 immunoprecipitates from PBL-L2 cells was identified as γTCR subunits by anti-Cγb serum, antisera against γTCR constant region peptides (see method section of FIG. 11A). (Called KD) was dissolved.

The γTCR protein in PBL-L2 associates noncovalently with the δTCR subunit, which is visualized as a weak iodinated protein in anti-CD3 immunoprecipitates analyzed under non-reducing conditions (Figure 11A, column 6, closed arrow). ). Weakly iodinated proteins show δ TCR subunits in PBL-L2 cells because they are not recognized as anti-Cγb serum (column 11A, column 8). It also shows the same SDS-flexibility change compared to the analysis under non-reducing and reducing conditions, as known as δTCR protein in IDP2 and PEER cells (see PCT International Publication NO. Wo 88/00209, published January 14, 1988). The δTCR protein was not visualized after reduction (Figure 11A, column 3) because it resided at 40 KD (see below) and then darkened with γδ TCR protein of similar size (open arrow).

This γTCR form is not only seen in normal peripheral blood T lymphocytes but is also observed in thymic-induced clone II cells (Figure 11D) and in T-leukemia cell culture MOLT-13 (Figure 11E). These three cell cultures showed different glycosylation, resulting in γTCR protein dipoles or MOLT observed in PBL-L2 (40KD and 44DK; column 11A FIG. 8) and clone II cells (40KD and 44DK; column 11A FIG. 8 column). Diffusion-labeled γTCR protein bands observed at −13 (40-46 KD; column 11E FIG. 6) were obtained. Two-dimensional gel analysis of MOLT-13γTCR protein bands (SDS-PAGE followed by unbalanced pH gradient electrophoresis (NEPHGE)) dissolves two equilibrium γTCR species (40KD and 44DK), of which 44KD γTCR species are further compared to 40KDγTCR species. Mannose (or hybrid) N-linked glycans were included. The γTCR subunits of these receptor complexes isolated from three different cell sources (peripheral blood, thymus and leukemia) thus exhibited 40KD cell surface species covalently associated with the δTCR counterpart chain.

For comparison to the γδ TCR type of PBL-L2 clone II and MOLT-13 cells, we tested a known form of IDP2 WM-14 cell culture. IDP2 cell cultures (PCT International Publication NO. Wo 88/00209, published January 14, 1988; Brenner et al., 1986, Nature 322: 145-149) are larger than those recognized as anti-Cγb iron (Figure 11B). 55-60 KD γTCR protein (called 55 KD). When anti-CD3 immunoprecipitates are tested in non-reducing conditions, it is clear that the IDP2 γTCR protein is noncovalently associated with its δTCR counterpart chain (column 11 in Figure 11B). Upon reduction, the δ TCR protein appears in SDS-PAGE mobility for a molecular weight of 40 KD (compared to the open arrows in column 11B, column 4, and the closed arrows in column 11B, column 2).

Contrary to the non-covalently integrated γTCR form, peripheral blood-derived T cell embryonic lines, WM-14, are anti-Cγ serum (Alarcon et al., 1987, Proc. Natl. Acad. Sci. USA 84: 3861-3865). It has a 70KD disulfide-linked TCRD dimer (column 11C, column 7), which is also recognized as anti-TCRδ1, a mAb against the δTCR subunit (column 11C, column 5C). Therefore, the γδ TCR heterodimer is represented as three γTCR proteins (referred to as 35KD, 40KD and 43KD) by analysis under reducing conditions.

As such, the CD3-associated complex of PBL-L2 clone II and MOLT-13 cells constitutes a novel γTCR heterodimer as compared to the known form because its TCRγ subunit is 40KD (in WM-14 cells). It constitutes a disulfide-bound Cγ1 infused γTCR protein, but not its counterpart chain (similar to 55KD Cγ2 encoded γTCR protein in IDP2 cells). To understand the molecular basis of this complex, more detailed structural analysis of the γTCR and δTCR subunits was performed as described below using MOLT-13 cell culture lines as in the examples.

8.2.2. Core polypeptide size of the MOL13 γTCR subunit

To determine the size of the γTCR core polypeptide of MOLT-13 cells (40KD γTCR glycoprotein and compare it to the size of PEER cells (55KD γTCR glycoprotein), the two cell cultures were ³⁵ S-methionine and ³⁵ S dissolved in Triton X-100. -Labeled with 15-part biosynthesis in the presence of cysteine and then immunoprecipitated with anti-Cγml, a monoclonal antibody that specifically recognizes the γTCR chain (see FIG. 13A, method section). Was subsequently digested with endoglycosidase H (Endo H) to remove unmature N-linked glycans The MOLT-13 γTCR polypeptide backbone had a relative molecular weight of 35KD (column 13A, column 8) and it was PEER γTCR core polypeptide (40KD; column 13A Figure 8) or IDP2 γTCR core polypeptide (40KD; PCT International Publication No. WO88 / 00209, published January 14, 1998) was 5KD smaller than MOLT-13 cells were 5KD smaller. IDP2 by size It can be concluded that the PEER γTCR core polypeptide is expressed as a unique γTCR core polypeptide, and only 5-11KD in post-translational process (40-46KD surface size) in some MOLT-13 γTCR cell surface glycoproteins Minus 35KD core size) in this case a relative molecular weight of 15-20KD can be calculated as a post-translational process for PEER and IDP2 γTCR glycoprotein (55-60KD surface size minus 40KD core size). Assuming that they are bound glycans and each glycan chain is calculated with a relative molecular weight of approximately 3KD, they indicate that 2 to 3 N-linked glycans are attached to the MOLT-13 γTCR protein, while on the other hand 5N-linked Polypeptides in glycan PEER and IDP2 cells Experiments using N-glycans with N-linked carbohydrates from cell surface γTCR proteins have been found in most post- It indicates that the reverse process is actually a combination N- glycans.

8.2.3. MOLT-13γTCR is 1st order

In order to understand the structure of the constant region gene fragment encoding the MOLT-13γTCR subunit, the sequence of cDNA clones corresponding to MOLT-13γTCR transcription was measured. γTCR cDNA clone, pTγ-1 (Dialynas et al., 1986, Proc. Natl, Acad. Sci. U. S. A. 83: 2619-23). One clone M13K was selected based on the size and limitation of the restriction map and displayed full length in γTCR transcription using the Vγ1.3 gene fragment (Lefranc et al., 1986, Cell 45: 237-246; Lefranc et al., 1986, Nature 319: 420-422; nomenclature based on Strauss et al., 1987, Science 237: 1217-1219; Quertermous et al., 1987, J. Immunol. 138: 26872690). The constant region sequence is the recently reported non-functional γTCR (Pellici et al., 1987, Science 287: 1051-1055) and two CII exon transcripts b and c (Lefranc et al., 1986, Proc. Natl. Acad Sci. USA 83: 9596-9600), found to be nearly identical to the sequence order of the Cγ2 genome (see Method section for more detailed calculations). This represents the first frame internal transcription that encodes a γTCR protein expressed on the cell surface using two CII exon transcriptions.

The imagined amino acid sequence of the cDNA clone shows a polypeptide backbone size of 34.8 KD which is in good agreement with the biochemical data described above. Surprisingly, six potential N-linked carbohydrate deposit sizes are encoded by transfer. Because biochemical data suggest that only 2 to 3 N-linked glycans are attached to the polypeptide chain, not using all potential positions is due to the use of the Cγ gene fragment (Krangel et al., 1987, Science 237: 64-67). Large (55 KD) non-disulfide-linked γTCR subunits in the γδ TCR form expressed in IDP2 and PEER cells have three CII exons, namely transcription a, transcription b and transcription c (Krangel et al., 1987, Science 237: 64). -67). Large (55 KD) non-disulfide-linked γTCR subunits in the γδ TCR form expressed in IDP2 and PEER cells have three CII exons, namely transcription a, transcription b and transcription c (Krangel et al., 1987, Science 237: 64). -67; Littman et al., 1987, Nature 326: 85-88), encoded as a Cγ2 gene fragment and therefore this γδ TCR form is referred to herein as Form 2abc. Therefore, the form characteristic of MOLT-13 cells is called form 2bc.

8.2.4. Preferred Cγ Gene Fragments Use

To determine the presence of these three γ, δTCR forms of newly isolated peripheral blood, we analyzed mononuclear cells taken from 10 healthy subjects using biochemical assays with mAb anti-TCRδ1 (see paragraph 6 above). Reactive with the described δ TCR constant region). This antibody reacts with the majority if all are not γ, δTCR lymphocytes. Representative results from this panel are shown in FIG. 15. In subject 1, the anti-TCRδ1 immunoprecipitates (analyzed under non-reducing conditions) were 70KD protein blood (form 1), two disulfide-linked γ, δTCR complexes and a wide 40KD protein blood (form 2bc) (Figure 15 2). Heat) indicates the presence of non-disulfide-linked γ, δ TCR complexes. This indicates that both Cγ1 and Cγ2 constant regions are used by these individual expressed γ, δ TCRs. However, the amount of form 2bc varied between individuals. Note the smaller fraction of Form 2bc in Subject 2 compared to Pizza 1 by comparing the strength of the 40KD protein bands in the two individuals (comparing column 2 of Subject 2 with column 2 of Subject 1). Even more shocking was that only disulfide-linked γ, δ TCR complexes could be detected in three mononuclear cells out of ten individuals tested even after long exposure of radiographic methods (see Subject 3). None of the individuals analyzed showed 55 KD bisulfide-linked γδ TCR complex (form 2abc) in peripheral blood.

8.2.5 Characterization of the δTCR subunit

In contrast to the shocking structural differences in the size and glycosylation of γTCR proteins, the δTCR subunits from other cell sources prove to be remarkably similar. Relative molecular weights of δTCR glycoproteins in MOLT-13 cells were measured directly at 40KD using anti-δTCR mAbs (column 4 of FIG. 12) to size δTCR glycoproteins of IDP2 cells (column 11 of FIG. 11B). Confirmed similar.

The cell surface I-labeled δTCR protein taken from MOLT-13 cells was also digested with N-glycanase to compare the δTCR polypeptide backbone size, asparagine-linked glycans (high mannose, hybrid and complexed-form; Tarentino et al., 1985, Biochem. 24: 4665-4671; Hirani et al., 1987, Anal. biochem. 162: 485-492). The δTCR core polypeptides of MOLT-13 cells have a relative molecular weight of 35KD (column 13B in FIG. 4B) similar to the δTCR backbone of IDP2 cells (35KD) (BAND ET AL., 1987, Science 238: 682-684).

Endoglycosidase H (Endo H, which removes only high mannose and certain hybrid N-glycans; Tarentino et al., 1974, J. Biol. Chem. 249: 811; Trimble and Maley, 1984, Anal. 141: 514-512), the cell surface I-labeled MOLT-13 δ TCR protein was digested, leaving a 2.5KD relative amount consistent with the presence of one carbohydrate component. Two potential N-glycan attachment sites in the δ TCR region (ㅗㅗ Hata, s., et al., 1987, Science 238: 678-682; Loh et al., 1987, Nature 330: 59-572) These data indicate that both are used but their glycans are treated differently, that is, one is treated with high mannose N-glycans (Endo H-resistant, N-glycanase folk). In contrast to the different amounts of N-linked carbohydrates to which the γTCR polypeptide chain is attached, the δTCR subunits expressed in PEER, IDP2 and MLOT-13 cells all had the same peptide core size and two N-linked glycans (Fig. 13B, Data not shown).

8.3. discussion

In this example, human γTCR glycoproteins in the form of three proteins were compared. Namely disulfide-linked 40KD γTCR protein (form 1), non-sulphide-linked 55KD γTCR protein (form 2abc) and non-sulphide-bound 40KD γTCR protein (form 2bc). All three forms are shown associated with the δTCR subunit. The complementary DNA sequence showing the first two γTCR forms was previously reported (krangel et al., 1987, Science 237: 64-67; Littman et al., 1987, Nature 326: 85-88). ΓTCR polypeptide form 1 ( A region of (in PBL-C1) is encoded by a Cγ1 gene fragment containing a single CII exon, while the γTCR polypeptide form 2abc (in IDP2 and PEER cells) is CII exon transcription a, transcription b and transcription c. Use a Cγ2 gene fragment comprising. The cDNA sequence corresponding to the γTCR chain of Form 2bc was found to include a Cγ2 gene fragment using only two CII exon electrons, transcription b and transcription c. Similarly, the genetic structure of the γTCR linker region of clones II and PBL-L2 (non-disulfide-linked, 40KD γTCR protein) also appears to be the same as the MOLT-13 structure measured excitation of form 2bc. Because the δ TCR constant region used is the same for all these forms (Hata, S., et., 1987, Science 238: 678-682; Loh, EY, et al., 1987, Nature 330: 569-572). A complete comparison of this structure of the three γTCR forms of was performed at this time (Fig. 16).

Two genotypes of Cγ2 polymorphism exist in humans (Lefranc et al., Nature 319: 420-422; Pellici et al., 1987, Science 237: 1051-1055). Two transcriptional forms (Action 2abc and Form 2bc) would be products of these different allelic forms. Currently, the allelic form of γTCR polypeptide has not been found in mice. We conclude that the amount of attached N-linked carbohydrates in which the dramatic difference in γTCR cell surface protein size between Form 2abc (55KD) and Form 2bc (40KD) most closely reflects the number of N-linked glycans. It is mainly measured by. Skeletal sizes of the IDP2 γTCR (form 2abc) and MOLT-13 γTCR (form 2bc) proteins were determined to be 40 KD and 35 KD, respectively, based on SDS-PAGE, and their estimated molecular weights of 36.6 KD calculated from the cDNA sequence. Corresponds well to 34.8 KD respectively. It is clear that the small differences in skeletal size, calculated mainly by one CII exon encoded peptide of 16 amino acids, contributed to the observed molecular weight differences between 55KD and 40KD non-disulfide-linked γTCR surface morphology but cannot be fully explained. . Form 2abc γTCR polypeptides carry five potential N-linked glycan attachments, all of which are likely to be used against MOLT-13 γTCR polypeptides carrying two to three N-linked glycans and having one additional potential attachment site. Has a location. The reason for this limited use of potential attachment sites is unknown, but may be due to the effect of CII exon encoded peptides on the structure of the γTCR protein. CII exon encoded peptides and their neighboring amino acids create a linker region between the plasma membrane and an immunoglobulin-like region. Their region includes most of the N-linked glycan attachment sites (FIG. 17). I believe that CII exon transcription measures protein morphology not only by measuring polypeptide backbone size, but also by creating an ability for disulfide-binding chains and also affecting the amount of carbohydrate attached.

Δ TCR of IDP2 (Hata et al., 1987, Science 238: 678-682), complementary DNAs, PEER (Loh et al., 1987, Nature 330: 569-572) and MOLT-13 cells were sequenced, variable and The same turned out to be the same except for the diversity / N-region that spacing between the constant region gene fragments. The δTCR protein in WM-4 cells has a relative molecular weight of 43 KD, which is described by the previously described δTCR protein (Borst et al., 1987, Nature 35: 683-688; Lanier et al., 1987, J. Exp. Med. 165). 1076-1094), but 3KD larger than other δTCR chains. Such 43KD δTCR protein may indicate the presence of additional N-linked glycosylation sites in different variable regions.

Comparable structural differences to those described for the δ TCR constant region fragment were observed for the αTCR and βTCR genes (Yoshikai et al., 1985, Nature 316: 837-840; Toyonaga et al., 1985, Proc. Natl. Acad Sci. USA 82: 8624-8628; Royer et al., 1984, J. Exp. Med. 160: 947-952; Kronenberg et al., 1985, Nature 313: 647-653). There is a structural similarity in the number of human CII exons repeating in length in the rat Cγ region, of which Cγ1, Cγ2 and Cγ3 constant regions inject 15, 10 and 33 amino acid linkage regions, respectively (Garman et al., 1986, Cell 45: 733-742; Iwamoto et al., 1986, J. Exp. Med. 163: 1203-1212). However, the linker region in rats reflects the difference in the size of the associated exons, not the overlapping use of exons as seen in human form 2abc γTCR and form 2bs γTCR. In addition, mouse γTCR is present only in disulfide-bonded forms, in contrast to two non-disulfide-linked human forms.

Importantly, the human γ and δ TCR forms do not appear to be used the same. In some individuals (selected for high% γ, δ TCR lymphocytes), the single form (form 1) predominates, suggesting that either positive selection occurs for this form and that there are also opposing choices in other γ, δ TCR forms. .

9. Three Reconstructed by Paired Pairs of Unique Transfected δTCR Chains with a Single δTCR Subunit

T cell receptor γ, δ genotype morphology

In the examples herein, the role of γTCR polypeptides in the formation of γδ heterodimers was investigated. We tested by transfecting γδ TCR complexes corresponding to three γTCR forms (form 1, 2abc and 2bc) with a single residual δTCR chain. The γδ heterodimer consisting of form 2bcγTCR covalently transfected with δTCR polypeptide into γTCR DNA encoding any of Form 1 or Form 2 of the γTCR polypeptide into the MOLT-13 cell culture. To express. It can express a heterodimer consisting of 2abcγTCR noncovalently associated with γTCR or Form 1γTCR noncovalently bound to δTCR along with transfected MOLT-13 cell culture γTCR characteristics. Moreover, glycosylation of transpeptated γTCR gene products was equivalent to glycosylation of these genes in their natural cell cultures. As such, the degree of glycosylation and the ability to form disulfide bonds are measured by the γTCR gene, but the use of γTCR constant region CII exons does not only measure the presence or absence of disulfide bonds between TCRγ and δ polypeptides, but also the γTCR chain. The amount of carbohydrates attached to the cells is also measured, and this amount depends largely on the size difference of the cell surface γTCR protein.

9.1. Materials and methods

9.1.1. Cell culture

One TCRγδ ⁺ T leukemia cell culture MOLT-13 (Hata, S., et al., 1987, Science 238: 678-682; Loh, EY, et al., 1987, Nature 330: 569-572) and peripheral Blood-derived TCRγδ ⁺ cell cultures PBL C1 (Brenner, MB, et al., 1987, Nature 325: 689-694) and IDP2 (Brenner, MB, et al., 1986, Nature 322: 145-149) Incubate as described previously.

9.1.2. Antibodies

The monoclonal antibody (mAb) used was as follows: anti-Leu-4 (anti-human CD3; IgG1) (Ledbetter, JA et al., 1981, J. Exp. Med. 153: 310-323), anti -TCRδ1 (anti-human TCRδ chain constant region; IgG1) (see Section 6; Band, H., et al., 1987, Science 238: 682-684), anti-Ti-γ (anti-Vγ2; IgG2a) ( Jitsukawa, S., al., 1987, J. Exp. Med. 166: 1192-1197), anti-Cγml (anti-human γTCR constant region; see Section 8.1.7), P3 (IgG1 secreted into P3X5 63Ag8 myeloma) (Koehler, G., and Milstein, C., 1975, Nature 256: 495-497) and 187.1 (anti-mouse k light chain specificity of mice (Yelton, DE, et al., 1981, Hybridoma 1: 5-) 11).

9.1.3 Isolation and Sequence of MOLT-13 δTCR cDNA Clone

Supplementary DNA prepared from MOLT-13 poly A ⁺ RNA in the delivery medium λgt10 (Huynh, et al., 1985, in DNA Cloning, ed.Glover, DM (IRL Press, Oxford), Volume 1, pp. 49-78) (cDNA) libraries were sieved by hybridization to P-labeled human δ TCR cDNA clone IDP20-240 / 38 (Hatd et al., 1987, Science 238: 678-682). Clones were selected for detailed analysis on the basis of size and limited restriction map. Dideoxy chain termination method using a T7 polymerase (Sequenace, United States Bichemical Corp.) (Poter et al., 1984, Proc. Natl. Acad. Sci. USA 81: 7161-7165) modified with nucleotide sequence ( Sanger et al., 1977, Proc. Nat l. Acad. Sci. USA 74: 5463-5467).

9.1.4. Construction of Expression Plasmids and Transfection

As outlined in FIG. 10B, γTCR cDNAs (PBL Cl.15 and IDP2.11r) (Krangel, MS, et al., 1987, Science 237: 64-67) were used to form a Friend spleen focal forming virus (SFFV). PFneo mammalian expression delivery media (Saito, T., et al., 1987, Nature 325: 125-130; Ohashi, P., et al., 1985, Nature 316) downstream of the long term repeat (LTR) : 606-609). Plasmid constructs were transfected into MOLT-13 cells by electroporation (Potter, H., et al., 1984, Proc. Natl. Acad. Sci. 81: 7161-7165). Transfects were selected and maintained in medium containing 2 mg / ml G4l8 (480 μg / mg solids by biopsy) and cloned with limited dilution.

9.1.5. Iodide and Immunoprecipitation

Lactoperoxidase, 3-[(3-colamidopropyl) dimethylammonio] 1-propanesulfonate (CHAPS; Sigma Chemical Co., St. Louls, MO), immunoprecipitation by various antibodies, non-equilibrium pH gradient Cell surface labeling by gel electrophoresis (NEPHGE) and SDS polyacrylamide gel electrophoresis (SDS-PAGE) dmf tkdydgksms ¹²⁵ I was performed as described (see Section 8.1.3, Brenner, MB, et al., 1986, Nature 322: 145-149, Brenner, MB, et al., 1987, Nature 325: 689-694). Specific immunoprecipitation was performed in 1 μg anti-Leu-4, 0.1 μl anti-TCRδ1 ascites or 1 μ1 P3 ascendant with 150 μl of 187.1 culture supernatant. For anti-T9-γA, 1 μl ascites was used without 187.1.

9.1.6. Biosynthetic Labeling

30 min the cells in exponential growth and expression in methionine-free system other medium and then for ³⁵ S- methionine for ³⁵ and 15 minutes at 37 ℃ pulse labeled with cis S- and others, and immunoprecipitation are wioe in sections 8.1.3 It was done as described. Immunoprecipitates were treated or mock-cultured with Endo-Glycosiase-H (Endo-H), separated by SDS-PAGE and visualized by fluorography (Bonner, WM and Laskey, RA, 1974, Eur. J. Biochem. 46: 83-88).

9.2. result

We studied the products obtained by the association of structurally distinctive γTCR gene products with a single δTCR protein to indicate the role of the γTCR gene, and in particular the TCR CII exon, in determining the structural differences between the various γTCR genotypes. For this purpose, MOLT-13, a T leukemia cell culture expressing a 40KD non-disulfide-linked γTCR polypeptide (form 2bc), is used as a receptor for γTCR chain cDNA clones corresponding to two other types of receptors (forms 1 and 2abc). It was. These γTCR chains are described in Figure 14 (form 2bc) and Krangel et al., (1987, Science 237: 64-67) (forms 1 and 2bc) in the complete sequence of cDNA clones and they are shown in Figure 18A. It is shown schematically.

9.2.1. The δTCR chain of a single functional group is present in the MOLT-13 cell culture line.

ΔTCR gene rearrangement studies of MOLT-13 cell cultures (Hata, S., et al., 1987, Science 238: 678-682) suggested that only monofunctional δTCR gene products were expressed in this cell culture water. . However, crosslinking-hybridization with the δTCR cDNA probe (Hata, S., et al., 1987, Science 238: 678-682) to directly indicate that transcription of mono-functional groups for δTCR is made in MOLT-13 cells. The cDNA clones were isolated from the MOLT-13 cDNA library prepared in λgo10 and the sequence of the selected cDNA clones was determined. This analysis showed that MOLT-13 cells rearranged into one functional group and expressed transcription corresponding to one rearranged δTCR gene. CDNA clones corresponding to rearrangements and δ TCR genes as functional groups are characterized by V (Vδ1), J (Jδ1 as described previously for IDP2 cell cultures (Hata, S., et al., 1987, Science 238: 678-682). ) And C gene fragment. However, MOLT-13 δ TCR cDNA clones use D fragments (MOLT-13 uses only Dδ2), inaccurate binding and N-region diversity in VD and DJ junctions (Hata, S., et., 1983, Science 240: 1541-1544) has a unique nucleotide sequence between the V and J gene fragments. MOLT-13 δ TCR cDNA also represents cysteine residues in the membrane junction region of certain gene fragments that can be used for disulfide binding to γTCR gene products using Cγ1 gene fragments. Although MOLT-13 cell cultures express non-disulfide-linked γ, δTCR receptors, the linker region of each base of its δTCR chain may be involved in non-disulfide-bonded or disulfide-bonded complexes of δTCR subunits. Open the possibility that

9.2.2. The γTCR gene product determines this type of receptor.

MOLT-13 cells transfected with γTCR cDNA constructs were transfected with Ml3.PBL C1γ (for MOLT-13 cells transfected with PBL C1-derived γTCR cDNA) and M13.IDP2γ (IDP2-derived γTCR cDNA). O) for MOLT-13 cells. Bulk transfected cell cultures and representative subclones derived from these cell cultures were analyzed by route blot analysis with γTCR (VJC) or Vγ2-specific cDNA probes. In addition to the residual 1.6 kb MOLT-13 γTCR transcription, a second γTCR transcription of about 1.8 kb (the size predicted for the onset γTCR transcription in the SFFV LTR of the expressed plasmid) was observed in transfect cultures and clones thereof. 1.8 kb transcription specifically hybridized with Vγ2 probe that does not cross-hybridize with Vγ1.3 is present in the residual MOLT-13 γTCR transcription and thus 1.8 kb transcription is performed using transfected γTCR cDNAs (Vγ2 fragments). ) Transcription.

Representative clones derived from each culture to biochemically characterize γTCR protein (s) expressed on the surface of transfects were identified as P3 (baseline), anti-leu-4 (anti-CD3), anti-TCRδ1 (anti-δTCR). ) Or immunoprecipitation of the surface iodinated cells as anti-Ti-γAmAbs. Specific γ, δTCR cells using the Vγ2 gene fragment as variable proteins of their γTCR chains of anti-Ti-γA (Jitsukawa. S., et al., 1987, J. Exp. Med. 166: 1192-1197). To be recognized. Not only transfected but also transfected MOLT-13 cells (FIGS. 19B and 19C) express the expected γTCR (40KD; see open arrow) and δTCR subunit (see asterisk). Note that the anti-Vγ2-specific mAb (anti-Ti-γA) fails the reaction of the residue with the MOLT-13 γTCR chain (columns 19A and 7A). The anti-CD3 immunoprecipitates of Ml3.PBL C1γ transfect cells show additional CD3-associated species (68KD) when tested in non-reducing conditions (see column 3 in column 19B, see arrow). PBL C1 cells (columns 3 and 4 in Figure 19D) and MOLT-13. In both PBL C1γ transfected cell cultures (columns 19B, 3 and 4), the 68KD complex yielded 40 and 36KD species upon reduction. For M13.PBL C1γ transfects, these bands are anti-Vγ2 immunity. It is clearly visible in the sediment. 19B see FIGS. 7 and 8 continuation arrows). These 40 and 36KD species represent differentially glycosylated γTCR polypeptides (Brenner, M. B., et al., 1987, Nature 325: 689-694). In these immunoprecipitates the δ TCR chain (40 KD reduced) co-moves with the 40 KD γTCR polypeptide and is therefore not visible (but see below).

Importantly, these experiments showed that disulfide-linked γ, δTCR heterodimers of transfected cell cultures in which the residue δTCR chain of MOLT-13 cell cultures, normally a portion of a non-disulfide-bonded complex, associates with the PBL C1 γTCR protein. To form. In contrast, IDP2-induced γTCR protein (55KD) in the M13.IDP2 transspectant formed a non-disulfide-bonded complex as the residue MOLT-13 δTCR chain (19C FIGS. 3 and 4). Immunoprecipitates carried out with anti-TCR δ1 mAb (specific for δTCR peptides) were transfected γTCR chains from these cell cultures as well as endogenous (columns 5 and 6 of Figures 19A, D and E). (Columns 5 and 6 in Figures 19B and C) were found to associate directly with δTCR. Anti-Ti-γA was 68KD disulfide-linked γ, δTCR heterodimer and M13.EDP2γ transfectant cells from M13.PBL C1 transfectant cells (columns 7 and 8 of FIG. 19B). And specifically immunoprecipitating the 55KD γTCR chain along with the 40KD δTCR chain from column 8) to confirm that the γTCR chain, which is part of this complex, corresponds to the transfected PBL C1 and IDP2-induced γTCR cDNAs, respectively.

To further characterize various γTCR proteins, two-dimensional (2D) gel analysis (NEPHGE and SDS-PAGE) of surface ¹²⁵ I-labeled cells was performed. The location of the CD3 component allows for comparison of γTCR species involved in the overlapping of 2D forms of the residual (MOLT-13) and transfected (PBL C1 or IDP2) γTCR chains. ΓTCR chains in immunoprecipitates from MOLT-13 cells were analyzed as two discontinuous parallel series of iodinated species (see also arrow open in Figure 20A). MOLT-13 cells transfected with PBL C1 or IDP2 TCR cDNAs showed a residual MOLT-13γTCR polypeptide family, but also PBL C1 (compare FIG. 20B and D; see asterisk in FIG. 20B) or IDP2 (in FIG. Comparison of FIGS. 20C and D; see closed arrows in FIG. 20C) shows one species with the same radiolabel as the γTCR polypeptide of the cells. Thus, 2D gel biochemical analysis confirmed that the transfected γTCR chain was expressed and treated similarly in MOLT-13 transspectant and in the cell culture poles PBL C1 and IDP2.

9.2.3. Polypeptide Skeleton Size of Transfected γTCR Chain Proteins

Substances immunoprecipitated from cells transfected with γTCR chains by means of metabolism of the peptide backbone were labeled by endoglycosidase-H. Immunoprecipitations performed with anti-CγM1 (specific to δTCR chains) demonstrated 35.5 and 34KD species in non-fected MOLT-13 cells (column 21, Figure 4) showing native MOLT-13 γTCR polypeptides. These two small polypeptides (see open arrow) correspond to the expected polypeptide core size of the MOLT-13 γTCR polypeptide, while the large polypeptide appears to represent a partially processed intermediate. In addition to these residual MOLT-13 γTCR polypeptides, 41KD polypeptides of deglycosylated size were immunoprecipitated with anti-CγM1 from M13.IDP2γ transspectant (see FIG. 21, column 8, sequential arrows). The size of these transspectant-specific γTCR polypeptides is in good agreement with the glycosylated IDP2 γTCR polypeptide cores measured beforehand in IDF2 cells (Brenner, M. B., et al., 1987, Nature 325: 689-694). As expected, M13.PBL C1γ transspectant cells represent additional γTCR proteins with glycosylated size of 32 KD (Figure 21, column 12, see continuation arrow), which has already reported γTCR for PBL C1 cell cultures. Compared well with polypeptide backbone size. This 32KD species is specifically immunoprecipitated by Vγ2-specific mAb, anti-Ti-γA (see Figure 21, column 3, sequential arrows) to allow for clear assignment of residual and transfected γTCR species in this cell culture. As such, the measured skeletal size of the transfected γTCR chains derived from IDP2 and PBL C1 cell culture lines is consistent with the skeletal size of these polypeptides in their underlying cell cultures. Compared to the γTCR polypeptide core sizes with these of the cell surface proteins, we infer that MOLT-13, IDP1 and PBL C1 derived γTCR chains involve 6, 14 and 8KD N-linked carbohydrates, respectively.

9.3. discussion

Three biochemically distinct forms of human γδ TCR subunit structures occur. In this study, we show that a single δTCR polypeptide can be combined with a γTCR chain representing each of the three receptor forms to reconstitute a suitable γ, δTCR heterodimer. The residual γTCR polypeptide of MOLT-13 (form 2bc) is 40KD and is associated covalently with the δTCR subunit. The δ TCR form corresponding to the disulfide-coupled receptor of PBL C1 (form 1) or the 55KD non-disulfide-coupled receptor of IDP2 cell culture (form 2abc) was reconstituted. This transfection study provides direct evidence that disulfide bonds are dictated by the use of γTCR fragment fragments because the residues of the MOLT-13 δTCR chain in the residue have PBL C1-induced disulfide-containing γTCR chains (Form 1). This has been shown to be involved in non-sulfide-coupled receptor complexes with bound receptors and IDP2-induced γTCR chains (form 2abc).

We found that a significant difference in size between 55KD (form 2abc) and 40KD (form 2bc) non-disulfide-linked γTCR polypeptides was attached to the γTCR polypeptide backbone (see section 8.2.2. Above). It is mainly due to the difference in the amount. As such, only 15KD of N-linked carbohydrates (form 2abc of IDP2 or PEER) or 15KD (form 2bc of MOLT-13) even have the same number (five each) of N-linked glycan receptor positions. It is attached to these γTCR polypeptides even if they are encoded by certain gene fragments used in both. These four N-linked glycosyls and positions are present in or around the CII exon-encoded linker region. In the examples herein, they are based on a comparison of the cell surface size of the mature cells with the peptide core size of the protein product of the transfected γTCR cDNA clone, the amount of N-linked carbohydrates attached to the transfected γTCR protein. It shows the same as seen in the culture. As such, the adaptation of two Cγ2 encoded protein fragments should be different enough to cause a dramatic difference in glycosyl. The main difference between these two forms of Cγ fragment is that the transcription s of the CII exon is present in the 55KD γTCR chain of form 2abc and not from the 40KD γTCR chain of form 2bc. As such, the presence or absence of such CII exon transcription may be a source of glycosylation differences that account for γTCR polypeptide size.

Structural changes in the human γδ TCR genotype are not precedent between T-cell receptors because these parallels are not observed for αβTCR.

10. T cell receptor rd complex incompatible with CD3 is identified in human endometeria glandura epithelium.

In the early stages of placental development, mononuclear cell infiltration is large in the helical arteries and endometrial glands in the mother's urinary tissue. This includes an unusual population of T linear cells of unknown function. Many hypervary germ layers express new forms of primary MHC antigens that are different from those expressed in most somatic cells. We tested panels of monoclonal antibodies on TCR δ heterodimers (γδ TCRs) of pregnant and non-pregnant uterus. Surprisingly, the γδ TCR complex was not detected in white blood cells but localized in the cytoplasm of cold epithelium in the endometrium from the pregnant uterus. This antibody also reacted with cold epithelium from the non-pregnant uterus and its reactivity was stronger in the secretion stage than in the proliferation stage of the menstrual cycle. However, γδ TCR did not associate with the CD3 complex as shown by testing immunoprecipitates using three different monoclonal antibodies against CD3 (OKT3, anti-leu-4, UCHT-1). αδTCR-positive cold epithelial cells did not react with monoclonal antibodies to αβTCR, which were also CD4- and CD8-negative. Moreover, Hansen epitelli cells lose 1-Heat MHC antigens in early pregnancy. These data suggest that endometrial cold cells with γδ TCR cause at least expressive changes in local regulation of gene expression.

11.Examples: Characterization of Antibodies of Human δT Cell Receptor Genes and AV _δ Specific Monoclones

We isolated δTCR gene rearranged from human γδ cell clone, AK119. Kδ probes were obtained from these DNA clones and used to measure the diversity of 13 human γ, δ T cell clones and 3γ, δ human T cell tumor cultures. All five different rearrangements were detected and they corresponded to rearrangements using 2 to 5 different X _δ genes. One particular rearrangement was always seen in human γ, δ T cells that reacted with mAb TCS δ1 (δ6TCAR-3). TCRδ1 also immunoprecipitated δTCR polypeptides from human γ, δ tumor cell cultures, and MOLT-13. We present evidence that the monoclonal antibody TCSδ1 recognizes an epitope encoded in AK119Vδ or together recognizes an epitope of the rearranged AK119 gene Vδ-Jδ gene.

11.1 Materials and Methods

11.1.1 Isolation and Sequence of AK119 δTCR cDNA Clone

cDNA libraries were generated from PBL T cell clone AK119 by the method of Gubler and Hoffmann (Gubler and Hoffman, 1983, Gene 25: 263). An amplified library of approximately 100,000 platelets was used with a P-labeled nick-translated Cδ probe isolated from a δTCR clone called 0-024 (Hata, S. et al., 1987, Science 2389: 678). Was selected. The longest hybridized cDNA clone (1.3kb clone C119δ 3) was selected for sequence analysis by the deoxy chain end method.

11.1.2. Cloning of Rearranged δTCR Genes

A DNA clone of the 3.5 kb genome containing the rearranged Vδ gene was obtained from AK119 cells as follows. EcoRI digested DNA was sized on preparative agar gel, ligated into gt10 and packaged and transfected into E. coli. Recombinant phage was selected with a ³² P-labeled nick translated 550 bp EcoRI fragment derived from cDNA clone, c119δ3. Rearranged clones called r119δ8 were isolated, including 0.8 kb HincII fragment (V region specificity) and 1 Kb HincII-EcoRI fragment (VJ region).

11.1.3 DNA Preparation

Fetuses and newborn thymus tissues were collected according to approved guidelines regarding patient rights and approvals. T cell clones were obtained from peripheral blood, rhombocytosis or cerebrospinal fluid (Hafler et al., 1985, Ann Neurol. 18: 451; Van de Griend et al., 1987, J. Immunol. 138: 1627). In all cases DNA was prepared by digesting with protein enzyme K in 1% sodium dodecyl sulfate followed by extraction with phenol / chloroform and ethanol immunoprecipitation.

11.1.4 Writing blot analysis

Genome DNA was digested with EcoRI and sized on 0.9% agarose gel and transferred to nitrocellulose. Hybridization was performed with ³² P-nick translated probes as described previously (Maniatis, 1982, Molecular Colning: A Laboratory Manual (Cold Spring Harbor Laboratory; Cold Spring Harbor, New York)).

11.1.5 Analysis of Cytopulouro Flowmeters

Mononuclear cells (PBMC) of peripheral blood of normal humans obtained from volunteers were isolated by sorting on a Ficoll gradient. PBMC and PBL T cell clones were stained with indirect immunofluorescence using TCSδ1mAB (referred to above as δTCAR-3; see section 7 above) and fluorescently-conjugated goat mouse IgG (Becton Dickinson) and in a fluorescently activated flow cytometer. Analyzed.

11.2. result

11.2.1. Diversity of δTCR Gene Rearrangements

We isolated a 1.3 kb δTCR cDNA clone whose T cell clone AK119 was named c119δ3 from the gt10 cDNA library using a Cδ probe. The order of the 5 ′ end of c119δ3 was determined and the use of the V _δ and Jδ genes already identified (Hata et al., 1987, Science 238: 678; Loh et al., 1987, Nature 330: 569). The arrangement order of the VJ junctions indicated that C119δ3 had in-frame VJ connections.

A 550 base pair (bp) EcoRI fragment (VJC probe) encoding all variability and linkages and portions of a region was isolated from c119δ3 and used for writing blot analysis of the DNA of the EcoRI digested genome from AK119. The probe detects germline 3.2 kb V _δ and lazy line 1.0 kb C _δ bands. AK119 is a 3.5kb band with additional rearrangement identical to the δTCR rearrangement of the supplement (described in Hata et al., 1987, Science 238: 678; Loh el al., 1987, Nature 330: 569) (rearrangement II in Fig. 22). Indicated. This 3.5 kb band was cloned from a library of EcoRI sized λgt10 genome using a VJC cDNA probe. Partial explanatory diagram of the δ TCR gene cloned and rearranged, referred to as r119δ 1, is shown in FIG. 17. Localization of variability and linkage regions was determined using J oligonucleotide probes and variable region specificity probes. A 1 kb VJ probe from r119δ1 was isolated by digestion with HincII and RcoRI enzymes (see also FIG. 17).

This VJ probe was used to measure the diversity of δTCR gene rearrangements in one panel of 13 human γδTCR positive T-cell clones and 3 human γ-δTCR positive tumor cell cultures. As shown in Figure 22, five or five common rearrangements of the IV side are seen in polyclonal neothymocyte samples (column 11). This rearrangement is representative of the rearrangements used by human γδ T cell clones. Only rearrangement II hybridizes to HincII to HincII V _δ . Although I do not know that all of these rearrangements represent VDJ but not DJ rearrangement, some of these must represent rearrangements of the new variable region for the J _δ gene fragment characterized above. This is because they express organoleptic δ TCR polypeptide on their cell surface. I have not ruled out the possibility that this new rearrangement represents a rearrangement of a single new V _δ gene for other J _δ genes still to be identified. Their data is consistent with the fact that there should be 2-5 variable region genes available for δTCR gene rearrangement.

11.2.2 Measuring Specificity of mAb TCSδ1

TCSδ1, previously expressed as δ TCAR-3, was generated by melting splenocytes from mice immunized with human tumor γδ fTCR cell culture MOLT-13 with rat myeloid culture. When used in the fluorescence activated cell sorter assay, TCSδ1 did not react with all human γδ T cells but only with a few. The results are shown in Table 1. There is a complete correlation between the use of the AK119 V _δ gene (rearrangement II) and its positive staining by TCSδ1. This data provides strong evidence that epitopes recognized as δTCSl are encoded in the combinatorial epitopes of the AK119Vδ or rearranged AK119 Vδ-Jδ genes.

TABLE 1

1. δ TCR rearrangement detected with a V-J probe numbered I-V in FIG. 22.

2. Although no lamellar line J _δ was detected, only one rearrangement was identified in each case.

3. New rearrangements are observed in neonatal and fetal thymic cells. This rearrangement is called rearrangement VI.

4. + means positive coloring and-means negative coloring. nd means not detected.

12. Deposition of Hybridomas

The following hybridoma cell cultures producing the monoclonal antibodies shown were deposited with ATCC and received the following accession numbers.

The present invention is not to be limited in scope by the deposited cell cultures, because the deposited examples are intended as an illustration of one feature of the invention and any cell cultures that are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will be apparent to those of ordinary skill in the art from the foregoing specification and accompanying drawings. Such changes should be included within the scope of the claims of the present invention.

Claims (26)

In the method for producing γ, δ, T cell antigen receptor heterodimer, a) can express a nucleic acid encoding a γT cell antigen receptor polypeptide, b) A cell that can be transfected with a nucleic acid encoding a γT cell antigen receptor polypeptide is such a cell under conditions such that both the γT cell antigen receptor polypeptide and the δT cell antigen receptor polypeptide are expressed by the cell and are capable of forming a heterodimer. Method for producing γ, δ, T cell antigen receptor heterodimer, characterized in that consisting of culturing. The method of claim 1, wherein the γT cell antigen receptor polypeptide consists of amino acids of a region of the γT cell antigen receptor polypeptide 2bc encoded by nucleotides 439 to 1008 in the nucleotide sequence as follows (FIG. 14).

The method of claim 1, wherein the γT cell antigen receptor polypeptide is 2bc having a first amino acid sequence for the variable region, N, binding region, and constant region in a nucleotide sequence as shown in FIG. 14.

The method of claim 1, wherein the γT cell antigen receptor polypeptide is a γT cell antigen receptor 2bc type having a molecular weight of 40,000. The method of claim 1, wherein the subunits of the heterodimer are disulfide linkages and the γT cell antigen receptor polypeptide is a γT cell antigen receptor polypeptide type 1 having a molecular weight of about 40,000 Daltons. The method of claim 1, wherein the γT cell antigen receptor polypeptide is a γT cell antigen receptor polypeptide type 2abc having a molecular weight of 55,000 Daltons. A method of producing a portion of a γδ T cell antigen receptor heterodimer, the method comprising: a) expressing a nucleic acid encoding at least a portion of the γ T cell antigen receptor polypeptide, wherein the portion of the γ T cell antigen receptor polypeptide is constant, variable ΓT cell antigen receptor polypeptide portion consisting of a region and a binding region, and b) culturing a cell capable of expressing a nucleic acid encoding at least a portion of the γT cell antigen receptor polypeptide, wherein a portion of the γT cell antigen receptor polypeptide is present. The portion consists of a portion of the γT cell antigen receptor polypeptide consisting of a constant region, a variable region, and a binding region, and gives an expression capable of expressing the two nucleic acids by the cell, wherein the portion encoding the portion of the γT cell antigen receptor polypeptide Nucleic acid is transfected into the cell Characterized in that the method. A method of expressing a portion of a γδ T cell antigen receptor heterodimer, the method comprising: a) expressing a nucleic acid encoding at least a portion of the γ T cell antigen receptor polypeptide, wherein the portion of the γ T cell antigen receptor polypeptide is constant, variable ΓT cell antigen receptor polypeptide portion consisting of a region and a binding region, and b) culturing a cell capable of expressing a nucleic acid encoding at least a portion of the γT cell antigen receptor polypeptide, wherein a portion of the γT cell antigen receptor polypeptide is present. The portion consists of a portion of a γT cell antigen receptor polypeptide consisting of a constant region, a variable region, and a binding region, and gives an indication to express two nucleic acids by the cell, wherein the portion encodes a portion of the γT cell antigen receptor polypeptide. Nucleic acid is transfected into the cell Characterized in that the method. 9. The γT cell antigen receptor polypeptide is of type 2bc having a molecular weight of about 40,000 Daltons, and constant and variable regions of amino acid sequences encoded by nucleic acids 439 to 1008 in the nucleotide sequence as shown in FIG. The method consists of.

A γT cell antigen receptor polypeptide selected from a population consisting of a constant region, a variable region and a binding region, and a γδ T cell antigen receptor linked to a δT cell antigen receptor polypeptide selected from a population consisting of a constant region, a variable region, a binding region and a diversity region, A heterodimer is a cell capable of expressing a portion of a γ, δT cell antigen receptor heterodimer that is not linked to a CD3 complex. The cell according to claim 10, wherein the cell is an endometrial microsomal epithelial cell. A γT cell antigen receptor polypeptide of a heterodimer is a transfected cell expressing a γδ T cell antigen receptor heterodimer, wherein the nucleic acid encoding the γT cell antigen receptor polypeptide is produced by transfection into a cell. The cell of claim 12, wherein the γT cell antigen receptor polypeptide has a molecular weight of 40,000 Daltons and a sequence consisting of a constant region and a variable region composed of amino acid sequences encoded by nucleotides 439 to 1008 in a nucleotide sequence as shown in FIG. 14. .

In a cell expressing a part of the γ, δT cell antigen receptor heterodimer, the γ, δT cell antigen receptor is selected from the group consisting of a constant region, a variable region and a binding region. Consisting of a δT cell antigen receptor polypeptide selected from a population consisting of a binding domain and a diversity domain, the heterodimer is not linked to the CD3 complex, and the γT cell antigen receptor polypeptide is a cell infected with a nucleic acid encoding a γT cell antigen receptor. Characterized in that it is produced within. The method of claim 1, wherein the cells are T cells. The method of claim 1, wherein the nucleic acid encoded by the γT cell antigen receptor polypeptide is selected from i) a nucleic acid consisting of one CII exon, ii) a nucleic acid consisting of two CII exons, and iii) a nucleic acid consisting of three CII exons. The method of claim 1, wherein the expressed γT cell antigen receptor polypeptide and the expressed δ T cell antigen receptor polypeptide are non-covalently linked. The method of claim 1, wherein the expressed γT cell antigen receptor polypeptide and the expressed δ T cell antigen receptor polypeptide are covalently linked. The method of claim 7, 8 or 9, wherein the cells are T cells. The nucleic acid encoding a portion of the γT cell antigen receptor polypeptide is selected from i) a nucleic acid consisting of a single CII exon, ii) a hexane consisting of two CII exons, and iii) a hexane consisting of three CII exons. How to be. The method of claim 7 or 8, wherein the γT cell antigen receptor and the δT cell antigen receptor are noncovalently linked. The method of claim 7 or 8, wherein the γT cell antigen receptor and the δT cell antigen receptor are covalently linked. A method of determining the relative use of a γT cell antigen receptor polypeptide in a human sample, the method comprising: a) quantifying the amount of γ type 2bc T cell antigen receptor polypeptide in a sample comprising the T cells of the subject, and b) in the sample of the subject. Quantifying the amount of γT cell antigen receptor polypeptide selected from the group consisting of 1γ polypeptide and 2abc γ polypeptide, and comparing the amounts quantified in steps a) and b) to determine relative use. Nucleic acid sequence of which the nucleic acid is a cDNA clone. Hexane vector consisting of cDNA clone according to claim 24. A cell comprising a hexane vector according to claim 25.

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